Selectivity in the Efflux of Glucuronides by Human Transporters: MRP4

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Selectivity in the efflux of glucuronides by human transporters MRP4 is highly active towards 4-methylumbelliferone and 1-naphthol glucuronides, while MRP3 exhibits stereoselective propranolol glucuronide transport Erkka Järvinen, Johanna Troberg, Heidi Kidron, and Moshe Finel Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00366 • Publication Date (Web): 29 Aug 2017 Downloaded from http://pubs.acs.org on August 30, 2017

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Molecular Pharmaceutics

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Selectivity in the efflux of glucuronides by human transporters

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MRP4 is highly active towards 4-methylumbelliferone and 1-naphthol glucuronides, while

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MRP3 exhibits stereoselective propranolol glucuronide transport

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Erkka Järvinen1, Johanna Troberg1, Heidi Kidron2 and Moshe Finel1*

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Biosciences, Faculty of Pharmacy, University of Helsinki, Finland

Division of Pharmaceutical Chemistry and Technology,

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Division of Pharmaceutical

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Corresponding Author:

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*Moshe Finel, Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy,

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University of Helsinki, 00014 University of Helsinki, Finland, tel. no. +358 50 4480748,

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[email protected]

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ACS Paragon Plus Environment


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Abstract

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Xenobiotic and endobiotic glucuronides, which are generated in hepatic and intestinal epithelium

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cells, are excreted via efflux transporters. Multidrug resistance proteins 2-4 (MRPs 2-4) and the

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breast cancer resistance protein (BCRP) are efflux transporters that are expressed in these

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polarized cells, on either the basolateral or apical membranes. Their localization, along with

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expression levels, affect the glucuronide excretion pathways. We have studied the transport of

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three planar cyclic glucuronides and glucuronides of the two propranolol enantiomers, by the

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vesicular transport assay, using vesicles from baculovirus-infected insect cells expressing human

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MRP2, MRP3, MRP4, or BCRP. The transport of estradiol-17β-glucuronide by recombinant

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MRP2-4 and BCRP, as demonstrated by kinetic values, were within the ranges previously

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reported. Our results revealed high transport rates and apparent affinity of MRP4 towards the

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glucuronides of 4-methylumbelliferone, 1-naphthol and 1-hydroxypyrene (Km values of 168, 13

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and 3 µM, respectively) in comparison to MRP3 (Km values of 278, 98 and 8 µM, respectively).

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MRP3 exhibited lower rates, but stereoselective transport of propranolol glucuronides, with

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higher affinity towards the R-enantiomer than the S-enantiomer (Km values 154 vs. 434 µM). The

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glucuronide of propranolol R-enantiomer was not significantly transported by either MRP2,

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MRP4 or BCRP. Of the tested small glucuronides in this study, BCRP transported only 1-

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hydroxypyrene glucuronide, at very high rates and high apparent affinity (Vmax and Km values of

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4400 pmol/mg/min and 11 µM). The transport activity of MRP2 with all the studied small

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glucuronides was relatively very low, even though it transported the reference compound,

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estradiol-17β-glucuronide, at high rate (Vmax = 3500 pmol/mg/min). Our results provide new

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information, at the molecular level, of efflux transport of the tested glucuronides, which could

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explain their disposition in vivo, as well as provide new tools for in vitro studies of MRP3,

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MRP4 and BCRP.

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Keywords:

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hydroxypyrene, 1-naphthol, MRP2/ABCC2, ABCG2/BCRP, MRP3/ABCC3, MRP4/ABCC4

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Abbreviations:

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1-hydroxypyrene glucuronide (1-HPG); 1-hydroxypyrene (1-HP); 1-naphthol glucuronide (1-

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NPG); 4-methylumbelliferone glucuronide or 7-hydroxy-4-methylcoumarin glucuronide (4-

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MUG); 4-methylumbelliferone (4-MU); ATP-binding cassette transporter (ABC); breast cancer

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resistance protein (BCRP); estradiol-17β-glucuronide (E2-17G); multidrug resistance protein

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(MRP); R-propranolol glucuronide (R-PRG); S-propranolol glucuronide (S-PRG); UDP-

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glucuronosyltransferase (UGT);

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1. Introduction

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Drugs, other xenobiotics and endobiotics are often excreted from the human body as metabolites

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that result from oxidation or conjugation reactions (phases I and II, respectively), or their

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combination.1 Conjugation with glucuronic acid, called glucuronidation, which is catalyzed by

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one or several of the UDP-glucuronosyltransferase (UGT) enzymes, is a prevalent metabolic

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pathway for many small xenobiotics.2 These conjugates, commonly called glucuronides, are more

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hydrophilic than their parent compounds and mostly do not cross cell membranes passively.3,4 As

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a result, they could accumulate inside cells to higher concentrations than in the blood circulation,

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unless they are efficiently transported across the membranes by efflux transporters. Glucuronides

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are actively excreted from cells through transporter proteins in the plasma membrane, such as the

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ATP-binding cassette (ABC) transporters. Thereby, these transporters can substantially affect the

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disposition of drugs and their metabolites, e.g. by contributing to enterohepatic circulation.

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The ABC transporter family contains several pharmacologically and pharmacokinetically

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relevant efflux transporters that transport drugs and their metabolites across cell membranes.5,6

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Multidrug resistance protein 2, 3 and 4 (MRPs 2-4 or ABCC2-ABCC4) and the breast cancer

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resistance protein (BCRP or ABCG2) transport anionic compounds such as glucuronides.3,6

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These transporters are expressed in the three main tissues that metabolize and excrete drugs and

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their metabolites, namely liver, intestine and kidney.5,6 MRP2 and BCRP are localized on apical

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membranes in hepatocytes and intestinal epithelia, excreting their substrates into bile and

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intestine, respectively. MRP3 is found on the basolateral membranes of the respective cells,

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contributing to systemic exposure of its substrates. Both, MRP2 and MRP4 are localized on

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apical membranes of proximal tubular cells in the kidney, contributing to active tubular secretion

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of organic anions, but in hepatocytes MRP4 is found on the basolateral membranes. Based on

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experiments in the intestinal cell line Caco-27 and Abcc4 knockout mice studies8, MRP4 is likely

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to be expressed also in the intestine and to be localized on the basolateral membrane of the

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epithelia.

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There is an emerging need to understand the cellular mechanisms that determine apical versus

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basolateral transport since these processes have large effects on the disposition of drugs and their

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metabolites.4,9 It has recently been described that glucuronides could interact with multiple

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organic anion transporters, including both uptake and efflux transporters.4 Some glucuronides are

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found in the blood circulation at higher levels than their parent molecules, suggesting an

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important role for basolateral membrane efflux transporters. Glucuronides are excreted from the

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circulation either extensively through urine, such as paracetamol10 and morphine glucuronides11,

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or via bile and feces, like raloxifene12 and ezetimibe glucuronides13. In some cases, such as the

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recently described pradigastat glucuronide, the glucuronidation is the main metabolic pathway

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and it is entirely excreted via bile and feces, while its plasma and urine levels are negligible,

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indicating major contribution of hepatic apical transporters.14 Several glucuronide conjugates are

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substrates for MRP2, MRP3, MRP4 and some for BCRP.3 However, only a few studies have

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directly compared glucuronide transport by these human transporters, attempting to characterize

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the differences and similarities between all of them.

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Propranolol is a beta blocker that is mostly used to treat cardiovascular diseases.15 It undergoes

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extensive metabolism in humans, where the major pathway is oxidation and subsequent

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glucuronidation of the oxidation metabolites.16 However, a fraction of the drug, about 15-20 %, is

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metabolized by direct glucuronidation. Administered propranolol is a mixture of two

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enantiomers, R and S, and in humans their stereoselective glucuronidation results in different

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amounts of R- and S-propranolol glucuronides.17,18 These glucuronides have five- to ten-fold

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higher plasma concentrations than propranolol and are excreted via urine, mostly by glomerular

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filtration.19

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One of the most widely used compounds for studying UGT activities is 4-methylumbelliferone

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(7-hydroxy-4-methylcoumarin, 4-MU).20 In addition, 4-MU has been used as a medication for

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bile therapy21 and hence comprehensive human pharmacokinetic and metabolism data are

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available for it.22 In humans, it is almost completely (95 %) metabolized to the glucuronide

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conjugate (4-MUG) and this glucuronide is rapidly eliminated via urine. In animals, previous

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studies with perfused livers from Abcc3 and Abcc4 knockout mice indicated a major role of

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Mrp3, but not Mrp4, in the hepatic disposition of 4-MUG.23

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The major phase I oxidative metabolites of the environmental pollutants naphthalene and pyrene

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are 1-naphthol and 1-hydroxypyrene, respectively.24 No comprehensive human pharmacokinetic

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or metabolism data are available for them, but based on in vitro experiments with human liver

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microsomes, both compounds are converted to the respective glucuronides at high rates.20,25 In

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addition, glucuronide metabolites of both compounds have been determined in human urine and,

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therefore, could indicate polycyclic aromatic hydrocarbon exposure.24

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In this study, we have examined the in vitro transport of 4-MUG, 1-naphthol glucuronide (1-

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NPG), 1-hydroxypyrene glucuronide (1-HPG), as well as R- and S-propranolol glucuronides (R-

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PRG and S-PRG) by vesicles containing the recombinant human efflux transporters MRP2,

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MRP3, MRP4 or BCRP, that were expressed in baculovirus infected insect cells. These five

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glucuronides (Fig. 1) are structurally related and are both in vivo and in vitro relevant model

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compounds, important issues when attempting to explore differences and similarities in transport

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activity between these human transporters. In combination with the known cellular location of the

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transporters, we aim to improve understanding of glucuronide metabolite disposition in humans.

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2. Materials and methods

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2.1 Chemicals and solvents

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Sodium salts of 1-NPG and E2-17G, 4-MUG and Ko143 hydrates and GSH were acquired from

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Sigma-Aldrich (St. Louis, MO, USA). The 1-HPG was previously synthesized in our laboratory25

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and for this study its purity was confirmed to be >99 %, based on fluorescence- and UV-HPLC

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analyses. Analytical grade solvents (acetonitrile, ammonium hydroxide water solution,

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dichloromethane, DMSO, formic acid and methanol) were also obtained from Sigma-Aldrich.

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Water soluble cholesterol/RAMEB (randomly substituted methyl-β-cyclodextrin) was from

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CycloLab (Budapest, Hungary). Complete Ultra (EDTA-free) and Pierce Mini (EDTA-free)

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protease inhibitor cocktail tablets were from Roche Diagnostics (Mannheim, Germany) and

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Thermo Fischer Scientific (Rockford, IL, USA), respectively. Mouse monoclonal antibodies

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against MRP3 (M3II-21) and MRP4 (F-6), as well as goat anti-mouse IgG-HRP conjugated

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antibody, were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). The water

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for assays and analyses was purified with Milli-Q water purification system and filtered through

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0.22 µM filter (Merck Millipore, Darmstadt, Germany). All other chemicals and reagents were

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obtained from known commercial suppliers.

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2.2 Enzyme-assisted synthesis of R-PRG and S-PRG

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A method was previously developed in our laboratory for enzyme-assisted synthesis of R-PRG

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and S-PRG, using recombinant UGTs that were expressed in our laboratory.26 For this study, the

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method was further optimized for enhanced yields. Two larger scale biosynthesis reactions were

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carried out separately, incubating UGT1A9 with S-propranolol and UGT1A10 with R-

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propranolol, in small conical flasks under conditions of gentle agitation at +37 °C for either 72 h

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(UGT1A9) or 48 h (UGT1A10). The S-PRG synthesis reaction was carried out in a total volume

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of 25 ml and contained 2 mM substrate, 28 mg total protein of UGT1A9 membrane homogenate,

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100 mM Tris pH 7.5, 10 mM MgCl2 and 4 mM uridine 5′-diphosphoglucuronic acid. The

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synthesis reaction for R-PRG contained 2 mM substrate, 50 mg total protein of UGT1A10

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membrane homogenate, 50 mM phosphate buffer pH 7.4, 10 mM MgCl2 and 4 mM uridine 5′-

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diphosphoglucuronic acid, in a total volume of 35 ml.

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The reactions were terminated by centrifugation to remove insoluble protein, followed by

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alkalization of the supernatant with sodium hydroxide and extracting it three times with equal

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amounts of dichloromethane. The water phases were acidified with formic acid and loaded onto

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SPE cartridges (Oasis HLB, 200 mg, 6 ml from Waters, Milford, MA, USA) that were

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subsequently washed with mixtures of 0-30 % methanol in water containing 2 % formic acid. The

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glucuronides were then eluted with 60 % methanol in 0.5 % ammonia water solution. The

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fractions were concentrated under vacuum, subjected to HPLC purification and the collected

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purified fractions containing the glucuronides were combined and freeze dried. The residues were

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solvated by a mixture of water, methanol and trace ammonia, transferred to pre-weighted vials,

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concentrated and subsequently kept under vacuum overnight to give ammonium salts of the

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glucuronides. The final yields were 24 % and 18 % of S-PRG and R-PRG, respectively. The

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identity of the glucuronides was confirmed by comparison of their retention times in HPLC with

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earlier synthesized and characterized standards.26 In addition, the chromatographic purity was

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characterized using fluorescence and UV-HPLC, which indicated it was 97 % and 99 % for S-

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PRG and R-PRG, respectively.

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2.3 Cloning of ABCC4 cDNA and construction of ABCC3 and ABCC4 cDNA containing

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expression vectors

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The ABCC4 cDNA was amplified from a commercial sample of normal human kidney cDNA,

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purchased from BioChain (Newark, CA, USA), using an oligonucleotide from the 5’-end that

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carries a BssH2 restriction site upstream the start codon and an oligonucleotide from the 3’-end

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with a Spe1 restriction site downstream the stop codon. The amplified PCR product, as well as

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the pFastBac1 (FB) vector, were digested with BssH2 and Spe1, and the purified DNA fragments

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were ligated. The resulting plasmid, MRP4-FB, was isolated from the few ampicillin-resistant

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colonies and the cDNA of the entire coding sequence of MRP4 within it was sequenced for

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verification and comparison to the GenBank accession no. NM_005845.

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The human ABCC3 cDNA (in pGEM plasmid) was a generous gift from Prof. Piet Borst, the

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Netherlands Cancer Institute, to the Centre for Drug Research, Division of Pharmaceutical

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Biosciences, Faculty of Pharmacy, University of Helsinki. To express MRP3 in baculovirus-

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infected insect cells, the cDNA of ABCC3 in pGEM was amplified as two separate fragments. A

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shorter 5’ region fragment, about 600 bp in length, was amplified using an oligonucleotide from

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the flank region to which an Mfe1 site was inserted and an oligonucleotide from the internal

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unique Sal1 site. The other, longer fragment, the 3’ region of the cDNA, was amplified using the

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complementary oligonucleotide from the internal Sal1site and an oligonucleotide from the 3’-end

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flank sequence with a new Spe1 site. Finally, the two amplified fragments, digested with either

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Mfe1 and Sal1 or Sal1 and Spe1, were co-ligated into the FB vector that was digested with

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EcoR1 and Spe1. The cDNA of the entire coding sequence of MRP3 in the MRP3-FB plasmid,

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isolated from the resulting colonies, was sequenced for verification and comparison to GenBank

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accession no. NM_003786.

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To express the human recombinant MRP3 and MRP4 in insect cells, baculovirus stocks were

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prepared and amplified according to the Bac-to-Bac methodology (Thermo Fisher Scientific,

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Waltham, MA, USA).

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2.4 Expression and vesicle preparation of MRP2, MRP3, MRP4 and BCRP

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Expression and vesicle preparation of the transporters were essentially done as previously

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described.27-29 Hence, Sf9 insect cells (from Spodoptera frugiperda) were cultured in HyClone

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SFX-insect media supplemented with 5 % fetal bovine serum, both from GE Healthcare Life

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Sciences (South Logan, UT, USA). Cells (1.6 million per ml) in suspension were infected with

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optimized amounts of recombinant baculovirus per transporter, or baculovirus containing no

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cDNA (control preparations, CtrlM for MRPs and CtrlB for BCRP, see below) and cultured for

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three days before harvesting and storage at -20 °C. For vesicle preparations, the harvested frozen

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cell pellets were thawed by suspending in cold buffer (50 mM Tris hydrochloride pH 7.0 and 300

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mM mannitol) containing protease inhibitors and incubated on ice for 45 min. The cells were

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then collected by centrifugation at 1 200 g for 10 min, followed by suspending the pellet in TME-

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buffer (50 mM Tris hydrochloride pH 7.0, 50 mM mannitol and 2 mM EGTA), homogenization

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by at least 40 strokes with a Dounce homogenizer with pestle B and incubation on ice for 60 min.

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Cell debris was then removed by centrifugation at 1 200 g for 10 min and the supernatant was

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subjected to ultracentrifugation, at 100 000 g, for 75 min to collect the membrane fractions. The

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resulting pellets were suspended in TME-buffer and passed 20 times through a 27-gauge needle

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to form the vesicles. The total protein concentration was then determined, by the Pierce BCA

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Protein Assay Kit (Thermo Scientific, Rockford, IL, USA), and the vesicles were diluted with

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TME-buffer to a final total protein concentration of 5 mg/ml, snap frozen in liquid nitrogen and

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stored at -80 °C until use.

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In the case of BCRP, vesicles and the specific control vesicles (CtrlB) for BCRP, were loaded

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with cholesterol after the ultracentrifugation by suspending the pellet in TME-buffer that also

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contained 2.5 mM cholesterol/RAMEB, incubating on ice for 20 min, diluting in TME-buffer

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(without cholesterol) and centrifuging again at 100 000 g for another 75 min, after which the

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vesicles were prepared as above. Cholesterol loading in BCRP vesicles was done to enhance the

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transport function of BCRP as reported and described previously.28-30

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2.5 SDS-PAGE and immunoblot analysis of MRP3 and MRP4

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For immunoblot analyses of the newly expressed (in our laboratory) MRP3 and MRP4, 15 µg of

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total vesicle protein containing either one of these transporter or control vesicles (CtrlM), were

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suspended in Laemmli sample buffer, supplemented with 2.5 % 2-mercaptoethanol. The samples

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were resolved by SDS electrophoresis in a pre-cast stain-free gradient 4-20 % polyacrylamide gel

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(BioRad Laboratories Hercules, CA, USA), alongside protein standard markers (Thermo Fischer

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Scientific Rockford, IL, USA) . The proteins were subsequently transferred onto nitrocellulose

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membrane and immunoblotted with the MRP3 (dilution of 1:1500) or MRP4 (dilution of 1:1000)

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specific primary monoclonal antibody, followed by the secondary antibody (HPR-conjugated-

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goat-anti-mouse, dilution of 1:3750) and detection.

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2.6 Vesicular transport assays

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The vesicular transport assay protocol was described earlier.27-29 The assays were conducted in

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polystyrene 96-multiwell plates, at a final volume of 75 µl. The transport assay mixtures

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contained 40 mM MOPS (adjusted to pH 7.0 with Tris HCl), 60 mM KCl, 6 mM MgCl2, 7 mM

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Tris HCl, 7 mM mannitol and 0.3 mM EGTA, as well as 40 µg total vesicles protein in each

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assay, either recombinant transporter containing vesicles or control vesicles. The stock solutions

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of 1-NPG, E2-17G, 4-MUG, 1-HPG, R-PRG, S-PRG and Ko143, were prepared in DMSO at a

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final concentration of 50 mM and stored at -20 °C. Subsequent compound dilutions were done in

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the assay buffer (MOPS-MgCl2-KCl) and the final assay concentration of DMSO was kept below

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1.4 % in all transport mixtures. The assay mixture, containing vesicles and substrate, was pre-

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incubated 10 min at 37 °C before initiating the assay by the addition of pre-warmed Mg-ATP to a

6

final concentration of 4 mM (+ATP samples) or of pre-warmed assay buffer (-ATP samples).

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The samples were incubated at 37°C and 500 rpm shaking for the indicated times (1-10 min, see

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the figure legends for details). The transport reactions were quenched by the addition of 200 µl

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cold buffer (40 mM MOPS adjusted to pH 7.0 with Tris HCl and 70 mM KCl) and were

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immediately transferred to opaque 96-multiwell 1.0 µm glass fiber filter plates (Merck Millipore,

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Darmstadt, Germany) under vacuum filtering. Each well was then washed five times with 200 µl

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of the quenching buffer. The studied compounds were finally eluted from the retained vesicles by

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applying 100 µl of 1:1 methanol:0.2 % formic acid water into each well, incubating for 30-60

14

min at room temperature with gentle shaking and subsequently centrifuging for two min at 3000

15

g. The resulting samples were subjected to HPLC quantification (see section 2.7).

16

Substrate-concentration dependent transport assays (kinetic analyses) were conducted as

17

described above, but using seven or eight different substrate concentrations. The incubation times

18

for the kinetic analyses are reported in the figure legends and were selected based on pre-

19

determined linear range of the transport for each compound and transporter combination

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(Supplementary figure S1). The ATP-dependent transport was calculated by subtracting the rates

21

in the absence of ATP (-ATP values) from the rates in the presence of ATP (+ATP values) and

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fitting the resulting values to either the Michaelis-Menten equation (v = Vmax [S] / ([S] + Km)) or

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the Hill equation (v = Vmax [S]h / (S50h + [S]h), where S is the substrate concentration, S50 is the

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concentration producing half-maximal reaction rate and h is the Hill coefficient, using least

2

squares fit. Goodness of the fit was inspected in each case by Eadie-Hofstee transformation of the

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experimental data and the coefficient of determination (R2).

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The single substrate concentration assays were performed in two independent experiments, while

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other data was from a single experiment. Each vesicular transport assay experiment, whether

6

single substrate concentration or kinetic assay, included three replicates of both +ATP and -ATP

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samples. The transport values are reported in pmol of substrate retained in the vesicles per total

8

vesicle protein per incubation time (pmol/mg/min). Means of the data were calculated and

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presented with ± SD as error bars. Statistical significance for the differences between +ATP and

10

–ATP values and the effect of Ko143, the BCRP inhibitor, were evaluated using the two-tailed

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nonparametric Mann-Whitney U-test. A p-value < 0.05 was considered to be statistically

12

significant. Determination of kinetic constants and 95 % confidence intervals (95 % CI) for them,

13

fitting of experimental data, statistics and the graphs were prepared using GraphPad Prism 5

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software (GraphPad Software, La Jolla, CA, USA).

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2.7 Analytical methods

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Quantification of 1-NPG, E2-17G, 4-MUG, 1-HPG, R-PRG and S-PRG transport was done using

17

HPLC, equipped with Poroshell 120 EC-C18 column (4.6 x 100 mm, 2.7 µm; Agilent

18

Technologies, Palo Alto, CA, USA) and a fluorescence detector. The column was kept at 40 °C

19

and the flow rate was 1 ml/min, using acetonitrile (B) and 0.1 % formic acid in water (A) as

20

eluents. Injection volumes were either 50-60 µl (1-NPG, E2-17G and 4-MUG), 2-10 µl (1-HPG),

21

or 20 µl (R/S-PRG). The following gradient programs were used to elute the compounds: 0-1 min

22

(28 % B), 1-3 min (28-45 % B) and 3-4 min (80 % B) for 1-NPG (at 2.6 min); 0-1 min (35 % B),

23

1-2.5 min (35-60 % B) and 2.5-3.5 min (80 % B) for E2-17G (at 2.2 min); 0-1 min (15 % B), 1-3

14


Page 15 of 48

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1

min (15-24 % B) and 3-4 min (80 % B) for 4-MUG (at 2.9 min); 0-1 min (45 % B), 1-2 min (45-

2

60 % B) and 2-3 min (90 % B) for 1-HPG (at 1.9 min); 0-1 min (28 % B), 1-2 min (28-60 % B),

3

2-2.5 min (85 %) for R-PRG and S-PRG (at 2.0 min and 2.2 min, respectively). All the

4

chromatographic methods included equilibrium time of at least 1.5 min at the initial

5

concentration of eluent B, before injecting the next sample. Excitation and emission wavelengths

6

were 293 nm and 324 nm for 1-NPG, 216 nm and 310 nm for E2-17G, 328 nm and 370 nm for 4-

7

MUG, 236 nm and 288 nm for 1-HPG, and 227 nm and 342 nm for R-PRG and S-PRG.

8

Linearity of quantification (R2 ≥ 0.99 for each analyte) was ensured at the entire range of the

9

measured sample concentrations. The quantification ranges were 2-1000 nM, 10-7000 nM, 2-

10

3000 nM, 1-8000 nM, 10-1000 nM and 10-2000 nM for 1-NPG, E2-17G, 4-MUG, 1-HPG, R-

11

PRG and S-PRG, respectively. All the standard samples were prepared in the same way as the

12

actual samples, namely in 100 µl of 1:1 methanol:0.2 % formic acid in water and filtered through

13

the filter plate before the quantification (see section 2.6).

14

15

15



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1

Page 16 of 48

3. Results

2

3

3.1 Transport of E2-17G by MRP2, MRP3, MRP4 and BCRP

4

The human efflux transporters MRP2 and BCRP were recently expressed in our laboratory and

5

their transport activity with suitable substrates, in vesicle preparations, was characterized.27-29

6

The expression of the human MRP3 and MRP4 in our laboratory is reported in this study for the

7

first time (see sections 2.3 and 2.4) and it was confirmed by immunoblot analyses of the

8

respective vesicles (see insets in Figs. 2A and 2B). As can be seen from the immunoblots,

9

correct-size proteins were detected by the specific monoclonal antibodies, while no such signals

10

were detected in the control vesicles (CtrlM).

11

To verify the activity of the above four recombinant transporters in our vesicle preparations, their

12

transport kinetics of E2-17G (a commonly used transporters’ substrate) were assayed and used as

13

positive controls for their activity (Figs. 2A-D). The MRP2 transport kinetics of E2-17G was best

14

described by the Hill equation with constants S50 = 124 µM and h = 2.3 (Fig. 2C, Table 1). The

15

E2-17G transport kinetics of MRP3, MRP4 and BCRP were all best described by the Michaelis-

16

Menten equation with Km values of 36 µM, 67 µM and 58 µM, respectively (Figs. 2A, 2B and

17

2D, Table 1). The results also suggest that the transport rates of E2-17G by MRP3 and MRP4 are

18

rather low in comparison to BCRP and, particularly, MRP2. Hence, our results are in line with

19

previously reported kinetic models, constants and transport rates for all these four transporters.30-

20

34

21

transport are in line with literature reports, the reported transport rates and Vmax values here and

22

below should be interpreted with care since we are not able to quantify the amount of functional

Although the relative (between transporters) and the absolute transport rate values of E2-17G

16


Page 17 of 48

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1

human transporter in the different membrane vesicle preparations. Nevertheless, the E2-17G

2

transport results of all the transporters indicate that they are functional and with rather

3

commonly-obtained level of active transporters. This allows us to compare the activity of

4

different transporters and to draw conclusions from it, particularly if the differences are large.

5

3.2 Transport of 1-NPG, 1-HPG and 4-MUG

6

Besides E2-17G, the glucuronides that were studied here are rather small and mostly planar (Fig.

7

1). Their transport by MRP2, MRP3 and MRP4 was first tested at two substrate concentrations,

8

10 µM (Figs. 3A-C) and 100 µM (Figs. 3D-F) in the presence and absence of ATP, to distinguish

9

between active and passive transport. At both substrate concentrations, MRP4 transported 4-

10

MUG and 1-NPG at much higher rates than MRP2 and MRP3. Transport of 1-HPG by MRP4 in

11

comparison to MRP2 and MRP3 was clearly higher at the 10 µM concentration. The addition of

12

5 mM GSH did not have any effect on the transport of these compounds by MRP2 (data not

13

shown). No significant ATP-dependent transport of the compounds was observed in the control

14

vesicles (CtrlM), with the exception of 10 µM 1-NPG (Fig. 3B).

15

The transport of 4-MUG, 1-NPG and 1-HPG by BCRP was also tested at the same two substrate

16

concentrations, 10 µM (Figs. 4A-C) and 100 µM (Figs. 4D-F). Since BCRP vesicles were

17

cholesterol-loaded (see section 2.4), similarly prepared cholesterol-loaded control vesicles (CtrlB)

18

were used as controls for the BCRP transport experiments. Although BCRP transported all the

19

compounds in an ATP-dependent manner, there were similar ATP-dependent transport rates of 4-

20

MUG and 1-NPG in the cholesterol-loaded control vesicles (Figs. 4A-B and 4D-E). In an attempt

21

to verify the contribution of BCRP to the transport of 1-NPG and 4-MUG, the BCRP inhibitor,

22

Ko143, was added at 1 µM, a concentration that was previously shown to fully inhibit BCRP

23

function in our vesicle preparations.29 Ko143 partly inhibited transport of 4-MUG and 1-NPG in

17



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Page 18 of 48

1

BCRP vesicles and the inhibition was significant at 10 µM substrate concentrations. In the case

2

of 1-NPG, but not 4-MUG, the inhibition was significant also at 100 µM substrate concentration

3

(Figs. 4A-E). However, it seems that the contribution of BCRP to the transport of 4-MUG and 1-

4

NPG is rather minor, even if there might exist high affinity transport of 4-MUG by BCRP (Fig.

5

4A). On the other hand, BCRP exhibited high rates of 1-HPG transport and this activity was only

6

minor, even if statistically significant, in the control vesicles (CtrlB) (Figs. 4C and 4F).

7

3.3 Transport of R-PRG and S-PRG

8

Glucuronides of the two enantiomers of propranolol were biosynthesized (see methods for the

9

synthesis and Fig. 1 for the structures) and the transport of R-PRG and S-PRG was studied under

10

the same conditions as 4-MUG, 1-NPG and 1-HPG transport. R-PRG was transported

11

significantly only by MRP3 (Figs. 5A and 5B). MRP3 also transported S-PRG, but at lower rates

12

than R-PRG (Figs. 5C and 5D). In addition to MRP3, low rates but still statistically significant

13

transport rates of S-PRG by MRP4 and BCRP, were observed at both 10 µM and 100 µM

14

substrate concentrations (Figs. 5C and 5D). The addition of 5 mM GSH had no effect on the

15

transport of these compounds by MRP2 (data not shown).

16

3.4 Transport kinetics of 4-MUG and 1-NPG by MRP3 and MRP4

17

Kinetic analyses of 4-MUG and 1-NPG transport by MRP3 and MRP4, the more active

18

transporters with these substrates, were carried out. Although MRP2 exhibited statistically

19

significant transport of both these glucuronides (Figs. 3A-E), its activity was too low to

20

characterize reliable concentration dependent transport. The ATP-dependent transport of 4-MUG

21

by both MRP3 and MRP4 could be well described by the Michaelis-Menten equation (Fig. 6A)

18


Page 19 of 48

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1

with a Michaelis-Menten constants (Km) of 278 µM and 168 µM for MRP3 and MRP4,

2

respectively (Table 1).

3

The ATP-dependent transport of 1-NPG by MRP3 and MRP4 also followed the Michaelis-

4

Menten model, but in this case, there were larger differences in the apparent affinity and transport

5

rates between them (Fig. 6B). While the Km value of MRP3 towards 1-NPG was 98 µM. The

6

corresponding value for MRP4 was almost ten-fold lower, i.e. a Km value of 13 µM, along with

7

very high transport rates of 1-NPG (Table 1).

8

3.5 Transport kinetics of 1-HPG by MRP3, MRP4 and BCRP

9

MRP3, MRP4 and BCRP exhibited high ATP-dependent transport of 1-HPG in the initial

10

screening assays, transport activity that remained statistically significant also in the presence of

11

higher substrate concentration (Figs. 3C, 3F, 4C and 4F). The ATP-dependent transport kinetics

12

of 1-HPG by these transporters was saturable at low concentrations and followed Michaelis-

13

Menten kinetics (Fig. 6C and 6D). The resulting Km values were 8.1 µM, 3.2 µM and 11 µM for

14

MRP3, MRP4 and BCRP, respectively (Table 1).

15

3.6 Kinetics of R-PRG and S-PRG transport by MRP3

16

Since MRP3 was the most active transporter in the screening experiment of R-PRG and S-PRG

17

transport (Fig. 5), kinetic assays were conducted only for this transporter. The ATP-dependent

18

MRP3 transport of both glucuronides followed Michaelis-Menten kinetics (Fig. 6E) and the

19

derived Km values were 154 µM and 434 µM for R-PRG and S-PRG, respectively (Table 1).

20

21

19



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1

Page 20 of 48

4. Discussion

2

3

The transport of 4-MUG, 1-NPG, 1-HPG, R-PRG and S-PRG have not yet been studied

4

comprehensively with expressed human transporters in vitro. In this work, we have tested their

5

transport by the human efflux transporters MRP2, MRP3, MRP4 and BCRP, and found high

6

activity and affinity of MRP4 towards 4-MUG, 1-NPG and 1-HPG (Figs. 3-6). MRP3 exhibited

7

moderate transport activity, while the activity of MRP2 was very low or negligible towards all

8

the five tested compounds, even though high transport activity of E2-17G was observed, which

9

demonstrates that the MRP2 that was used in this study is fully active (Fig. 2C). In addition, only

10

MRP3 transported R-propranolol glucuronide at significant rates and was more active in S-

11

propranolol glucuronide transport than MRP4 and BCRP (Figs. 5 and 6D). The transport of 1-

12

HPG by BCRP was at high rate and affinity (Figs. 4C and 4F), while the Ko143-sensitive

13

transport of 1-NPG and 4-MUG by BCRP was low.

14

Presently, we have no method to compare the amounts of functional transporters in each

15

preparation, and thus the Vmax values may differ somewhat among preparations of even the same

16

transporter in the same system. Still, this variation is unlikely to significantly influence the

17

findings and the results from this work are further validated by the E2-17G transport results of the

18

different transporters (Fig. 2 and Table 1). The high rate and typical sigmoidal kinetic of E2-17G

19

transport by MRP2 (Fig. 2C and Table 1) is a particularly good example that no transport, or low

20

transport activity of other test compounds, such as 4-MUG or 1-NPG, by the same transporter

21

means that we are studying a poorly expressed transporter.

20


Page 21 of 48

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1

It may be added here that the results also reveal new data on the tested transporters that is not

2

affected by the ratio in expression levels between different transporters. This is the comparison in

3

the transport of the different compound by the same transporters. For example, the finding that

4

BCRP poorly transports 4-MUG and 1-NPG, but transports 1-HPG at very high rates (Fig. 4),

5

whereas MRP4 transports both 1-NPG and 1-HPG at rather similar rates (Fig. 3). Another

6

example is the stereoselectivity of MRP3 in propranolol-glucuronides transport. Higher or lower

7

expression levels of the transporter would have affected the transport rates of these drug

8

metabolites, but not the ratio between them, or the apparent affinity towards them.

9

4.1 Transport of 4-MUG

10

The main human metabolite of 4-MU, 4-MUG, is an interesting compound from the point of

11

view of this study due to the availability of comprehensive human in vivo pharmacokinetic data,

12

as well as animal studies findings. The parent compound, 4-MU, is almost completely

13

metabolized to its glucuronide in humans and the systemic clearance of 4-MU is very high.22

14

Most of 4-MUG is excreted via urine during the first five hours after 4-MU administration,

15

indicating lack of extensive enterohepatic circulation. In addition, the renal clearance of 4-MUG

16

exceeds the glomerular filtration rate more than three-fold, indicating active secretion of this

17

metabolite in the kidney by apical transporters and, perhaps, some extent of glucuronidation in

18

kidney followed by secretion into urine.

19

Our results (Figs. 3A, 3D, 4A and 4D) could indicate, along with the human in vivo findings,

20

negligible or minimal excretion of 4-MUG into bile by apical transporters in liver (MRP2 and

21

BCRP) and major contribution of basolateral transporters (MRP3 and MRP4) to the excretion of

22

4-MUG from the intestine and liver to the blood circulation. Furthermore, MRP4 is likely to play

23

a dual role in 4-MUG excretion. In the liver and intestine, where it is located on basolateral

21



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Page 22 of 48

1

membranes, MRP4 contributes to systemic excretion, while in kidney MRP4 affects the active

2

secretion of 4-MUG on apical membranes of proximal tubule cells. Interestingly, the peak plasma

3

concentration of 4-MUG could reach 30-60 µM22 and because intracellular concentrations are

4

probably higher, the observed Km values of this study (Table 1, 280 µM and 170 µM for MRP3

5

and MRP4, respectively) and the tested concentrations (Fig. 3, 10 µM and 100 µM) are relevant

6

in vivo values.

7

Results from animal studies on the transport of 4-MUG revealed similarities, as well as

8

differences, from our findings with human transporters, including apparent differences between

9

mice and rats. In perfused mice livers, the basolateral efflux of 4-MUG was about ten-fold higher

10

than its apical efflux and knockout of Mrp2 or Bcrp led to half reduced biliary excretion without

11

affecting the basolateral excretion of the glucuronide.35 In further studies, it was shown that

12

knockout of Abcc3, but not Abcc4, substantially affected the basolateral excretion of 4-MUG in

13

perfused mice livers, leading to about ten-fold reduced basolateral clearance and two-fold higher

14

intracellular accumulation of the glucuronide.23 On the other hand, it was showed in experiments

15

with everted mouse intestinal sacs that efflux of 4-MUG is higher into the apical compartment

16

than the basolateral side, along the intestine, when 4-MU was administered.36 However, when

17

everted intestinal sacs were prepared from Abcc3-/- mice, the basolateral efflux of 4-MUG was

18

substantially reduced. In rat, on the other hand, it was shown that 4-MUG was preferentially

19

excreted into bile in perfused livers, and that this excretion was reduced to minimal in rat livers

20

lacking Mrp2.37 In addition, in a study of isolated perfused rat kidneys, 4-MUG was

21

preferentially excreted to the perfusate rather than to the urine, but the urinary excretion was

22

four-fold higher than glomerular filtration rate.38

22


Page 23 of 48

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1

The findings from animal studies could be divided into two groups, mice results and rat results.

2

While 4-MUG is primarily excreted by transporters on the basolateral membrane in liver and

3

intestine in both humans and mice, it is excreted apically (i.e. into bile) in rats. The mice results

4

and our findings in this study agree on the role of MRP3 in 4-MUG transport, but disagree on the

5

contribution of MRP4. Whether the latter difference is due to difference in substrate specificity

6

between the mouse Mrp4 and the human MRP4, or originates from low expression level(s) of

7

that transporter in the rodent liver, remains to be explored. In any case, animal experiments

8

should be interpreted with care when building assumptions and models about the activity of the

9

human efflux transporters and their substrate specificities.

10

It should be added that in the human cell line Caco-2, the intracellularly generated 4-MUG was

11

excreted at more than five-fold higher amounts to the basolateral side, in comparison to the apical

12

side.39 This result indicates significant contribution of basolateral efflux transporters such as

13

MRP3 and MRP4, which would be in line with our findings of the high activity of human MRP3

14

and, especially MRP4, in 4-MUG transport (Figs. 3A, 3D and 6A).

15

The high activity of human MRP4 and MRP3 in 4-MUG transport was not unexpected. A similar

16

compound, 7-hydroxycoumarin glucuronide, was previously shown to be transported at high rate

17

and affinity by MRP4 and at a rather high rate by MRP3 when another in vitro system was used,

18

membrane vesicles from transporter-transfected human embryonic kidney 293 cells.40 In

19

addition, 4-MUG inhibited human MRP3 in vitro with a Ki-value of 105 µM41, a result that is in

20

range with our result that the Km value of MRP3 in 4-MUG transport is 270 µM (Table 1).

21

4.2 Transport of 1-NPG

23



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Page 24 of 48

1

In humans, 1-NPG is formed from 1-naphthol20, an oxidation product of naphthalene42. After

2

exposure to naphthalene, 1-NPG is found in human urine and it is recognized as a biomarker for

3

environmental polycyclic aromatic hydrocarbon exposure.24,42 No comprehensive human

4

pharmacokinetic data is available for 1-NPG or other naphthalene metabolites. However, 1-NPG,

5

as 4-MUG, has been studied extensively in different animal studies, including perfused livers,

6

kidneys and intestine.

7

In rats that were administered with 1-naphthol intravenously, it was found that more than 80 % of

8

the formed 1-NPG was excreted in urine.43 Similar findings were obtained in mice, where >70 %

9

of the orally administered 1-naphthol was excreted via urine and half of this consisted of 1-

10

NPG.44 In perfused rat intestine experiments, when 1-naphthol was administered, 80-90 % of 1-

11

NPG was excreted to the basolateral side.45-47 In addition, it was found in rats that the renal

12

clearance of 1-NPG exceeded more than five-fold glomerular filtration and it was not changed in

13

the strain that lacked Mrp2, indicating major contribution of renal apical transporters such as

14

Mrp4.47 This, in line with our human efflux transporter results (Figs. 3B, 3E, 4B, 4E and 6B),

15

could indicate that hepatic and intestinal basolateral transporters, such as MRP3/Mrp3 and

16

MRP4/Mrp4, as well as renal apical transporters, such as MRP4/Mrp4, play important roles in 1-

17

NPG disposition in vivo. On the other hand, in rat livers it was shown, using Mrp2 deletion

18

strain, that some bile excretion exists and knockout of this apical transporter abolished it.47

19

Human originated Caco-2 cells excreted 1-NPG, formed from 1-naphthol, into the basolateral

20

compartment at several fold higher amounts than to the apical compartment.48 In addition, it was

21

found in vesicle assays, that the transport activity of human MRP4 was substantially inhibited by

22

1-NPG.49 Overall these findings are in line with our new results (Figs. 3B, 3E and 6B), according

23

to which 1-NPG is a good substrate for the human MRP3 and, particularly, MRP4 due to its low

24


Page 25 of 48

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1

Km value for this transporter (Table 1). Hence, both transporters are likely to be involved in the

2

excretion of 1-NPG from human tissues in vivo, such as intestine, liver and kidney.

3

The above results also suggest that 1-NPG could serve as a good substrate for in vitro assays of

4

MRP4.

5

4.3 Transport of 1-HPG

6

The main metabolite found in human urine after exposure to pyrene is 1-HPG.50 One-

7

hydroxypyrene (1-HP), and especially 1-HPG, are considered as biomarkers for environmental

8

polycyclic aromatic hydrocarbon exposure, similarly to 1-naphthol and its glucuronide.24 No

9

comprehensive in vivo data on human metabolism or excretion of 1-HPG are available, but data

10

from several animal studies are present.

11

In rats, when pyrene was administered orally or parenterally, 1-HP was excreted via both urine

12

and feces of which the latter predominated.51-53 In bile-cannulated rats, biliary excretion of 1-HP

13

was three- to five-fold higher than urine excretion.51,52 Most of the biliary and urinary 1-HP was

14

in conjugated forms, such as 1-HPG, whereas practically all the fecal 1-HP was unconjugated.51,

15

53

16

high, but declined towards the large intestine, probably due to hydrolysis of the glucuronide by

17

gut bacteria. This suggests that the rat apical transporters, such as Mrp2 and Bcrp, transported 1-

18

HPG from the liver and intestine.53

19

In polarized Caco-2 cells, pyrene was administered to the monolayer of cells and 1-HPG was

20

analyzed from both the basolateral and apical compartments.54 Unfortunately, the amounts of

21

formed glucuronide conjugate in these cells was very small in comparison to the sulfate

22

conjugate of 1-HP. Nevertheless, a substantially higher fraction, around six-fold, of 1-HPG was

Nevertheless, it was found that along the rat small intestine the amount of 1-HPG was very

25



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Page 26 of 48

1

excreted into the apical than basolateral compartment. In addition, the apical excretion was

2

inhibited significantly by the rather specific BCRP inhibitor, Ko143. Hence, our results on 1-

3

HPG transport (Figs. 4C, 4F and 6C) agree with findings from both rat studies and human

4

originating Caco-2 cell line. Their outcome is that BCRP/Bcrp probably plays a major role in the

5

tissue excretion of 1-HPG, particularly in the intestine and liver. In addition, MRP3/Mrp3 and

6

MRP4/Mrp4 (Figs. 3C, 3F, 6C) could contribute to systemic exposure and subsequent urinary

7

excretion of 1-HPG from the intestine and liver.

8

4.4 Transport of propranolol glucuronides

9

Although propranolol glucuronides are not the main metabolites for propranolol, they are

10

interesting compounds because in humans they exist in the blood circulation at several fold

11

higher concentrations than propranolol, and are subsequently excreted into urine.16,17 Excretion of

12

the propranolol glucuronides is practically completed within 24 hours following propranolol

13

administration16 and there is no indication of fecal excretion of any drug-related material15. This

14

suggests that the excretion of propranolol glucuronides from metabolizing tissues (liver and

15

intestine) is to the blood circulation.

16

Our results with the expressed human efflux transporters (Figs. 5 and 6D) are somehow in line

17

with these in vivo human findings; propranolol glucuronides are mainly excreted via basolateral

18

transporters, such as MRP3, and no substantially apical excretion in liver and subsequent

19

enterohepatic circulation exists. Our results are also supported by the finding that in patients with

20

renal insufficiency there is around 20-fold accumulation of propranolol glucuronides in

21

comparison to healthy people, which indicates no other major excretion pathway for these

22

glucuronides than excretion into urine.55 In addition, there is no active tubular secretion of

23

propranolol glucuronides in kidney17,19

26


Page 27 of 48

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1

The finding here, that MRP3 transports R-PRG with higher affinity than S-PRG (Fig. 6D and

2

Table 1), is interesting but not exceptional among the MRPs. It was previously reported that the

3

inhibition efficiency of MRP4 by nonsteroidal anti-inflammatory drug glucuronides could vary

4

by up to 20-fold between two enantiomers of the same drug.56 Propranolol undergoes

5

stereoselective glucuronidation in humans and more S-PRG is excreted via urine than R-PRG.18

6

However, it was found that the excretion rate into urine18 and the plasma clearance of R-PRG17

7

were higher in comparison to S-PRG. Nonetheless, it is difficult to predict the effect of

8

stereoselective transport by MRP3 on the disposition of S-PRG and R-PRG. In addition,

9

glucuronidation of propranolol in the human intestine could result in different enantiomeric ratio

10

of the glucuronides in comparison to the metabolism in liver and thus affect their excretion rate.26

11

In conclusion, the human MRP4 and MRP3 transport small planar glucuronides such as 1-NPG,

12

4-MUG and 1-HPG. MRP3 was the major transporter of R-PRG and S-PRG of which the R-

13

enantiomer was transported at higher apparent affinity. On the other hand, MRP2 transported the

14

five tested glucuronides very weakly or not at all, whereas BCRP-mediated transport of 1-NPG,

15

4-MUG, R-PRG and S-PRG was low or negligible, while its transport of 1-HPG was at high

16

affinity and capacity. Our results may help to explain in vivo findings on the disposition of these

17

compounds, as well as the functions and differences of the human efflux transporters MRP2,

18

MRP3, MRP4 and BCRP that are involved in the transport of glucuronide conjugates in human

19

tissues.

20

21

Acknowledgments

27



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Page 28 of 48

1

We would like to thank Johanna Mosorin, Noora Sjöstedt and Feng Deng for skillful help in the

2

cloning, expression and transporter vesicle preparations.

3

The funding of the University of Helsinki Doctoral Program in Drug Research, Sigrid Juselius

4

Foundation (grant no. 4704583) and the Academy of Finland (grants no. 12600101 and 1292779)

5

are acknowledged.

6

7

Conflict of Interest

8

The authors declare no conflict of interests.

9

10 11

Supporting Information. Fig. S1 Linearity of transport versus time for all the transportersubstrate combinations that were included in the kinetic analyses (Figs. 1 and 6).

28


Page 29 of 48

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7 cell monolayers. Biochem. Pharmacol. 2002, 63, 123-128.

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coumarin metabolite 7-hydroxycoumarin glucuronide is mediated via multidrug resistance-

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associated proteins 3 and 4. Drug Metab. Dispos. 2012, 40, 1076-1079.

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41. Akita, H.; Suzuki, H.; Hirohashi, T.; Takikawa, H.; Sugiyama, Y. Transport activity of

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42. Preuss, R.; Angerer, J.; Drexler, H. Naphthalene - An environmental and occupational

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contribution to excretory metabolism. J. Pharmacol. Methods 1985, 13, 9-35.

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44. Chern, W.H. and Dauterman, W.C. Studies on the metabolism and excretion of 1-naphthol, 1-

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45. Koster, A.S. and Noordhoek, J. Glucuronidation in isolated perfused rat intestinal segments

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after mucosal and serosal administration of 1-naphthol. J. Pharmacol. Exp. Ther. 1983, 226, 533-

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538.

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intestine: The influence of 1-naphthol concentration, perfusate flow and noradrenaline. Naunyn-

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Schmiedeberg's Arch. Pharmacol. 1989, 340, 239-245.

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47. de Vries, M.H.; Redegeld, F.A.M.; Koster, A.S.; Noordhoek, J.; de Haan, J.G.; Oude

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Elferink, R.P.J.; Jansen, P.L.M. Hepatic, intestinal and renal transport of 1-naphthol-ß- d-

10

glucuronide

in

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11

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48. Siissalo, S.; Laine, L.; Tolonen, A.; Kaukonen, A.M.; Finel, M.; Hirvonen, J. Caco-2 cell

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49. Van Aubel, R.A.M.H.; Smeets, P.H.E.; Peters, J.G.P.; Bindels, R.J.M.; Russel, F.G.M. The

16

MRP4/ABCC4 gene encodes a novel apical organic anion transporter in human kidney proximal

17

tubules: Putative efflux pump for urinary cAMP and cGMP. J. Am. Soc. Nephrol. 2002, 13, 595-

18

603.

19

50. Strickland, P.T.; Kang, D.; Bowman, E.D.; Fitzwilliam, A.; Downing, T.E.; Rothman, N.;

20

Groopman, J.D.; Weston, A. Identification of 1-hydroxypyrene glucuronide as a major pyrene

21

metabolite in human urine by synchronous fluorescence spectroscopy and gas chromatography-

22

mass spectrometry. Carcinogenesis 1994, 15, 483-487.

35


hyperbilirubinemia.

Naunyn-


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51. Bouchard, M. and Viau, C. Urinary and biliary excretion kinetics of 1-hydroxypyrene

2

following intravenous and oral administration of pyrene in rats. Toxicology 1998, 127, 69-84.

3

52. Viau, C.; Bouchard, M.; Carrier, G.; Brunet, R.; Krishnan, K. The toxicokinetics of pyrene

4

and its metabolites in rats. Toxicol. Lett. 1999, 108, 201-207.

5

53. Saengtienchai, A.; Ikenaka, Y.; Darwish, W.S.; Nakayama, S.M.M.; Mizukawa, H.; Ishizuka,

6

M. Characterization and tissue distribution of conjugated metabolites of pyrene in the rat. J. Vet.

7

Med. Sci. 2015, 77, 1261-1267.

8

54. Hessel, S.; Lampen, A.; Seidel, A. Polycyclic aromatic hydrocarbons in food - Efflux of the

9

conjugated biomarker 1-hydroxypyrene is mediated by Breast Cancer Resistance Protein

10

(ABCG2) in human intestinal Caco-2 cells. Food Chem. Toxicol. 2013, 62, 797-804.

11

55. Stone, W.J. and Walle, T. Massive propranolol metabolite retention during maintenance

12

hemodialysis. Clin. Pharmacol. Ther. 1980, 28, 449-455.

13

56. Kawase, A.; Yamamoto, T.; Egashira, S.; Iwaki, M. Stereoselective inhibition of

14

methotrexate excretion by glucuronides of nonsteroidal anti-inflammatory drugs via multidrug

15

resistance proteins 2 and 4. J. Pharmacol. Exp. Ther. 2016, 356, 366-374. 74.

16

17

18

19

20 21

36


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1

Figure legends

2

Fig. 1. Structures of the glucuronide compounds.

3

Chemical structures of the six glucuronides that were used as substrates for the efflux transporters

4

in this study. The glucuronide conjugate moiety is represented as Gluc.

5

Fig. 2. Estradiol-17β-glucuronide transport.

6

ATP-dependent transport kinetics of E2-17G by the expressed human MRP3 (A), MRP4 (B),

7

MRP2 (C) and BCRP (D). The assays were carried out using 40 µg total vesicle protein in each

8

case and the incubation times were either 3 min (MRP3 and MRP4), 5 min (MRP2), or 6 min

9

(BCRP), before quenching the transport assay. The transport values, after subtraction of transport

10

without ATP, were fitted to either the Michaelis-Menten equation (MRP3, MRP4 and BCRP) or

11

the Hill equation (MRP2) and the derived kinetic constants, of single experiments containing

12

triplicate samples, are presented in Table 1. The solid lines represent fitting to the kinetic model.

13

Immunoblot analyses of MRP3 and MRP4 are shown, as insets, in panels A and B, respectively.

14

In these analyses 15 µg of total vesicle protein was subjected to SDS-PAGE and the transporters

15

were detected with specific monoclonal antibodies (see section 2.5 for details). Control vesicles

16

(CtrlM) were used as negative controls for the immunoblot analyses.

17

Fig. 3. Transport of 4-MUG, 1-NPG and 1-HPG by MRP2, MRP3 and MRP4.

18

The transport of 4-MUG (A and D), 1-NPG (B and E) and 1-HPG (C and F) by MRP2, MRP3

19

and MRP4 was studied at substrate concentrations of 10 µM (A-C) and 100 µM (D-F). The total

20

vesicular protein amount was 40 µg in each sample and the incubation times with the test

21

compounds were 5 min, in either the presence (closed bars) or absence (open bars) of ATP. Data

22

is presented as means ± SD from two independent experiments in triplicate samples. * denotes

37



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Page 38 of 48

1

statistical significant differences (p < 0.05 in the nonparametric Mann-Whitney U-test) between

2

+ATP and the respective –ATP values. Control vesicles (CtrlM) did not contain any recombinant

3

human transporter.

4

Fig. 4. Transport of 4-MUG, 1-NPG and 1-HPG by BCRP.

5

The transport of 4-MUG (A and D), 1-NPG (B and E) and 1-HPG (C and F) by BCRP was

6

studied at substrate concentrations of 10 µM (A-C) and 100 µM (D-F). The total vesicular protein

7

amount was 40 µg in each sample and incubation times with the test compounds were 5 min, in

8

either the presence (closed bars) or absence (open bars) of ATP. Both the BCRP-containing

9

vesicles and the control vesicles (CtrlB) were loaded with cholesterol (see section 2.4). The

10

transport of 1-NPG and 4-MUG by BCRP and CtrlB vesicles were also measured in the presence

11

of 1 µM Ko143 (a BCRP inhibitor). Data is presented as means ± SD from two independent

12

experiments in triplicate samples. * denotes statistical significant difference (p ≤ 0.05 in the

13

nonparametric Mann-Whitney U-test) between +ATP and the respective –ATP values. # denotes

14

statistical significant differences (p < 0.05 in the nonparametric Mann-Whitney U-test) between

15

+ATP values in the presence and absence of the inhibitor.

16

Fig. 5. Transport of R-PRG and S-PRG by MRP2, MRP3, MRP4 and BCRP.

17

The transport of R-PRG (A and B) and S-PRG (C and D) by MRP2, MRP3, MRP4 and BCRP

18

was studied at substrate concentrations of 10 µM (A and C) and 100 µM (B and D). The assay

19

conditions were as described above in the legends to Fig. 3 (MRP2, MRP3 and MRP4) and Fig. 4

20

(BCRP). Data is presented as means ± SD from two independent experiments. * denotes

21

statistical significant differences (p ≤ 0.05 in the nonparametric Mann-Whitney U-test) between

22

+ATP and the respective –ATP values.

38


Page 39 of 48

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1

Fig. 6. Transport kinetics of 4-MUG, 1-NPG, 1-HPG and propranolol-glucuronides

2

ATP-dependent transport kinetics of 4-MUG (A) and 1-NPG (B) by MRP3 and MRP4, 1-HPG

3

(C) by MRP3 and BCRP, 1-HPG by MRP4 (D) and MRP3 transport of R- and S-PRG (E) were

4

studied in single experiments containing triplicate samples. The transport values, after subtraction

5

of transport without ATP, were fitted to the Michaelis-Menten equation and the derived kinetic

6

constants are presented in Table 1. The experimental data are reported as means ± SD and the

7

lines represent fitting to the kinetic model. The amount of total vesicle protein in each case was

8

40 µg and the incubation times in the transport assays were either 1 min (MRP4 with 1-NPG and

9

1-HPG and BCRP with 1-HPG), 3 min (MRP4 with 4-MUG and MRP3 with 1-HPG), 5 min

10

(MRP3 with 1-NPG and 4-MUG), or 10 min (MRP3 with both propranolol-glucuronides).

11

39



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1

Page 40 of 48

Table 1. Kinetic constants of the tested transporters and substrates.

Transporter

Km

Vmax

µM (95 % CI)

pmol/mg/min (95 % CI)

2

R2

4-MUG MRP3 MRP4

278 (205-350) 168 (147-189)

191 (163-218) 778 (730-826)

0.96 0.98

192 (161-223) 1110 (1050-1160)

0.80 0.74

481 (451-512) 1510 (1370-1640) 4430 (4250-4600)

0.94 0.96 0.98

110 (101-119)

0.97

118 (89-146)

0.90

3520 (3280-3730)

0.99

163 (134-192) 243 (208-278) 678 (636-720)

0.80 0.90 0.98

1-NPG MRP3 MRP4

98 (58-138) 13 (9-16) 1-HPG

MRP3 MRP4 BCRP

8.1 (6.3-9.9) 3.2 (2.8-4.2) 11 (9-12) R-PRG

MRP3

154 (127-182) S-PRG

MRP3

434 (240-629) E2-17G

MRP2 MRP3 MRP4 BCRP

S50a =124 (113-135) ha = 2.3 (2.0-2.6) 36 (20-52) 67 (46-87) 58 (49-66)

3 4

The experimental data from Figs. 2 and 6 were fitted using the Michaelis-Menten equation, with

5

the exception of E2-17G transport by MRP2, where the Hill equation was used. The derived

6

kinetic constants of the best fits are reported, with confidence intervals (95 % CI) for the fitting

7

of the curves, in the parentheses.

40


Page 41 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

a

2

both in the Hill equation.

S50 is the concentration producing the half-maximal reaction rate and h is the Hill coefficient,

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

41



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1-hydroxypyrene glucuronide (1-HPG)

4-methylumbelliferone glucuronide (4-MUG)

1-naphthol glucuronide (1-NPG)

estradiol-17βglucuronide (E2-17G)

S-propranolol glucuronide (S-PRG)

R-propranolol glucuronide (R-PRG)

glucuronic acid

Gluc =

FIGURE 1.


Page 42 of 48

Page 43 of 48

B. MRP4

200

kDa 250 -

140

130 100 -

70 55 -

0

60 120 [E2-17G], µM

175

MRP2

200

kDa 250 -

140

130 100 -

70

55 -

0

D. BCRP

3600 2400 1200

0

E2-17G transport (pmol/min/mg)

MRP3

100 225 [E2-17G], µM

350




1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 A. 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36C. 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


60 120 [E2-17G], µM

175

60 120 [E2-17G], µM

175

600 400 200

0

FIGURE 2.



80

D. 4-MUG, 100 µM

*

transport (pmol/mg/min)

60 40 20

*

*

100

*

*

M

tr l C

R P4 M

R P3 M

M 200

*

100

* M

tr l C

M

R P4

tr l

R P2

M

0

C

R P4 M

R P3 M

C. 1-HPG, 10 µM 600

300

M

*

0

*

R P3

* *

400

M

40


*

20

R P2

M

tr l C

R P4 M

R P3 M

R P2 M 400 200 60

R P2

200

E. 1-NPG, 100 µM

B. 1-NPG, 10 µM

M

*

300

0

0

F. 1-HPG, 100 µM transport (pmol/mg/min)

*

400

* 200

*

3000 2000

*

*

1000

FIGURE 3.


M

C tr l

R P4 M

R P3 M

M

C tr l

R P4 M

M

M

R P3

0

R P2

0

4000

R P2


400

M


A. 4-MUG, 10 µM


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 44 of 48

Page 45 of 48

D. 4-MUG, 100 µM

*

40

*#

20

*

*

*

* *

100

*

*

*#

0


*#

3

B

C

+K o1 4

tr l

43

tr l

B

P+ R

C

BC

C

40

*

K

BC

B

tr l

R 60

20

o1

R

+K o1 4

P

3

B

tr l

o1 K P+

C

P R BC BC

400 300 200

*

*#

100

*

*

+K o1 4

3

B

tr l C

tr l

B

C

43 P+

R

F. 1-HPG, 100 µM transport (pmol/mg/min)

*

800 400

*

200

K

BC

B

C. 1-HPG, 10 µM

o1

R

+K o1 4

P

3

B

tr l C

tr l C

BC

R

P+

K

BC

o1

R

P

43

0

BC

0

4000 3000

*

2000 1000

FIGURE 4.


B

C tr l

P

B

tr l C

BC

R

P

0

R


200

E. 1-NPG, 100 µM

B. 1-NPG, 10 µM

1000

300

0

43

0

400

BC

60



A. 4-MUG, 10 µM


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



C. S-PRG, 10 µM

60


40

*

20

20

*

*

*

B

P R

C

BC

tr l

M

tr l

R P4

C

R P3

M

M

M

D. S-PRG, 100 µM transport (pmol/mg/min)

400 300 200

*

100

R P2

B

P R

C

BC

tr l

M

tr l

R P4

C

R P3

M

M

300 200

*

100

*

*

FIGURE 5.


B

tr l

C

P R BC

M

tr l

R P4

C

R P3

M

M

B

tr l

C

P R BC

M

tr l

R P4

C

M

M

R P3

0

R P2

0

400

R P2

M

B. R-PRG, 100 µM

M

40

0

R P2

0

60

M


A. R-PRG, 10 µM


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 46 of 48


PRG transport (pmol/min/mg)

1-HPG transport (pmol/min/mg)

0

250 500 [PRG], µM

B. 1-NPG

1-NPG transport (pmol/min/mg)

4-MUG transport (pmol/min/mg)

1 2 3 4 5 6 7 8 9 10A. 4-MUG 11 12 13 600 MRP4 14 15 16 17 400 18 19 20 200 21 MRP3 22 23 24 25 0 140 280 400 26 27 [4-MUG], µM 28 29 30 31C. 1-HPG 32 33 4500 34 35 36 BCRP 37 3000 38 39 40 41 1500 42 MRP3 43 44 45 0 25 50 75 46 47 [1-HPG], µM 48 49E. MRP3 and PRG 50 51 52 100 53 R-PRG 54 55 S-PRG 70 56 57 58 59 35 60

1300

MRP4

900 450 MRP3

0

140 280 [1-NPG], µM

400

D. MRP4 and 1-HPG

1-HPG transport (pmol/min/mg)

Page 47 of 48

1500 1000 500 0

0

700

FIGURE 6. ACS Paragon Plus Environment

5 10 [1-HPG], µM

15


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

membrane vesicle transport assay

ADP

R/S

ATP MRP2/3/4 and BCRP

For Table of Contents Only


Page 48 of 48

Selectivity in the Efflux of Glucuronides by Human Transporters: MRP4

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