multidrug resistance-associated protein 4 (mrp4/abcc4...

1521-009X/43/10/1430–1440$25.00 http://dx.doi.org/10.1124/dmd.115.065953DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 43:1430–1440, October 2015Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics

Multidrug Resistance-Associated Protein 4 (MRP4/ABCC4) ControlsEfflux Transport of Hesperetin Sulfates in Sulfotransferase1A3–Overexpressing Human Embryonic Kidney 293 Cells

Hua Sun, Xiao Wang, Xiaotong Zhou, Danyi Lu, Zhiguo Ma, and Baojian Wu

Division of Pharmaceutics, College of Pharmacy (H.S., X.Z., D.L., Z.M., B.W.) and Guangzhou Jinan Biomedicine Research andDevelopment Center, Jinan University, Guangzhou, China (X.W.)

Received June 15, 2015; accepted July 29, 2015

ABSTRACT

Sulfonation is an important metabolic pathway for hesperetin. How-ever, the mechanisms for the cellular disposition of hesperetin and itssulfate metabolites are not fully established. In this study, dispositionof hesperetin via the sulfonation pathway was investigated usinghuman embryonic kidney (HEK) 293 cells overexpressing sulfotrans-ferase 1A3. Two monosulfates, hesperetin-39-O-sulfate (H-39-S) andhesperetin-7-O-sulfate (H-7-S), were rapidly generated and excretedinto the extracellular compartment upon incubation of the cells withhesperetin. Regiospecific sulfonation of hesperetin by the cell lysatefollowed the substrate inhibition kinetics (Vmax = 0.66 nmol/min permg, Km = 12.9 mM, and Ksi= 58.1 mM for H-39-S; Vmax = 0.29 nmol/minpermg,Km = 14.8mM, andKsi= 49.1mM for H-7-S). The pan–multidrugresistance-associated protein (MRP) inhibitor MK-571 at 20 mMessentially abolished cellular excretion of both H-39-S and H-7-S

(the excretion activities were only 6% of the control), whereas thebreast cancer resistance protein–selective inhibitor Ko143 had noeffects on sulfate excretion. In addition, knockdown of MRP4 led toa substantial reduction (>47.1%;P < 0.01) in sulfate excretion. Further,H-39-S and H-7-S were good substrates for transport by MRP4according to the vesicular transport assay. Moreover, sulfonation ofhesperetin and excretion of its metabolites were well characterizedby a two-compartment pharmacokinetic model that integrated druguptake and sulfonationwithMRP4-mediated sulfate excretion. In con-clusion, the exporter MRP4 controlled efflux transport of hesperetinsulfates in HEK293 cells. Due to significant expression in variousorgans/tissues (including the liver and kidney), MRP4 should bea determining factor for the elimination and body distribution ofhesperetin sulfates.

Introduction

Phase II metabolism refers to various conjugation reactions, whereina polar moiety (e.g., sulfonate and glucuronic acid) is conjugated to thesubstrates. In general, phase II metabolism increases water solubility ofthe parent drug, thereby facilitating drug inactivation and elimination.Since more and more new drug entities are metabolized directly by phaseII enzymes, phase II metabolism is becoming increasingly important indrug discovery and development (Rowland et al., 2013). Cytosolicsulfotransferases (SULTs) are a family of enzymes that catalyze thesulfonation (or sulfation) reaction (i.e., addition of a sulfonate group tothe substrates), a type of phase II reaction (Klaassen and Boles, 1997).The sulfonation reaction represents an important mechanism in activityregulation and elimination of numerous endobiotics and xenobiotics,including dietary polyphenols (e.g., flavonoids) (Chapman et al., 2004;Allali-Hassani et al., 2007). Human SULTs (with a total of 14 enzymes)are divided into four families, namely, SULT1, SULT2, SULT4, and

SULT6 (Blanchard et al., 2004; Freimuth et al., 2004). Enzymes ofSULT1 and SULT2 families, with abundant expression in the liver andintestine, play a dominant role in catalyzing sulfonation reactions (Allali-Hassani et al., 2007; Teubner et al., 2007; Riches et al., 2009).Hesperetin (49-methoxy-39,5,7-trihydroxyflavanone) is a main and

widespread citrus flavonoid that shows versatile health benefits, such aschemopreventive, cardioprotective, and neuroprotective effects (Mantheyet al., 2001; Benavente-García and Castillo, 2008; Hwang and Yen,2008; Hwang et al., 2012). The beneficial effects of hesperetin onhealth are mainly attributed to its antioxidant, anti-inflammatory, andsignaling properties (Hwang et al., 2012; Roohbakhsh et al., 2015).Although showing favorable membrane permeability, hesperetin isconcerned with limited oral bioavailability (Kanaze et al., 2007;Kobayashi et al., 2008). One of the main causes for the poor absorptionof hesperetin is extensive phase II metabolism (i.e., glucuronidation andsulfonation) in the intestine and liver (Silberberg et al., 2006; Takumiet al., 2012). Hence, inhibition of metabolism appears to be an effectivestrategy to improve the bioavailability of hesperetin (Brand et al., 2010b).Consistent with the critical roles of glucuronidation and sulfonation in the

first-pass clearance of hesperetin, an array of UDP-glucuronosyltransferase(UGT) enzymes (i.e., UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A8,UGT1A9, UGT1A10, UGT2B4, UGT2B7, and UGT2B15) and multiplesulfotransferase (SULT) enzymes (including SULT1A1, SULT1A3, and

This work was supported by the Young Scientist Special Projects inBiotechnological Pharmaceutical Field of 863 Program [Grant 2015AA020916]and the National Natural Science Foundation of China [Grant 81373496].

H.S. and X.W. contributed equally to this workdx.doi.org/10.1124/dmd.115.065953.

ABBREVIATIONS: BCRP, breast cancer resistance protein; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; HEK, humanembryonic kidney; H-39-S, hesperetin-39-O-sulfate; H-7-S, hesperetin-7-O-sulfate; Km, Michaelis-Menten constant; Ksi, substrate inhibitionconstant; MRP, multidrug resistance-associated protein; PCR, polymerase chain reaction; qPCR, quantitative polymerase chain reaction; S1,hesperetin-39-O-sulfate; S2, hesperetin-7-O-sulfate; shRNA, short-hairpin RNA; SULT, sulfotransferase; UGT, UDP-glucuronosyltransferase; UPLC,ultra performance liquid chromatography; Vmax, maximal velocity.

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SULT2A1) are actively involved in conjugating hesperetin (Brand et al.,2010a). In addition, hesperetin conjugates are found to be the maincirculating metabolites in humans and rodents, confirming the impor-tance of conjugative metabolism in hesperetin disposition (Matsumotoet al., 2004; Takumi et al., 2012; Yamamoto et al., 2013). Further, thehesperetin conjugates (e.g., hesperetin-7-O-glucuronide) most likelyretain the biologic activities of the parent compound (Yamamoto et al.,2013). Hence, it is essential to elucidate the disposition mechanisms forthese hydrophilic metabolites.Transport of generated sulfates out of cells is an essential step in

compound elimination via the sulfonation pathway. Due to the polarnature, the sulfate conjugates cannot be transported by the passivediffusion mechanism. Transcellular transport of sulfate conjugates isfacilitated by membrane transporters (Zamek-Gliszczynski et al.,2006a,b, 2011). Investigations on the disposition of several drugs(i.e., acetaminophen, 4-methylumbelliferone, and ethinylestradiol) inrodents have revealed that the efflux transporters breast cancer re-sistance protein (BCRP) and/or multidrug resistance-associated protein(MRP) are significant contributors to the excretion of sulfate metabolites(Zamek-Gliszczynski et al., 2006a,b, 2011). Further, the study of Brandet al. (2008) has shown that BCRP is involved in apical excretion ofhesperetin conjugates in Caco-2 cells. However, there is no informationregarding the MRP transporters for efflux of hesperetin sulfates.MRP4/ABCC4 is the fourth member of the MRP family transporters

(also known as the C subfamily of ATP-binding cassette transporters)and is expressed in various organs/tissues, such as the brain, liver,and kidney (Russel et al., 2008). MRP4 mediates efflux transport ofnumerous xenobiotics/drugs, including antiviral, cardiovascular, andantibiotic drugs (Russel et al., 2008). Hence, like other transporters,MRP4 plays an important role in the elimination and body distributionof drugs (Giacomini et al., 2010; DeGorter et al., 2012). Furthermore,MRP4 is involved in cellular communication and signaling because ofits marked ability to transport many signaling molecules, such as cyclicnucleotides, eicosanoids, and urates (Russel et al., 2008).Phase II conjugates are the main circulating and potentially active

metabolites of hesperetin. However, the disposition mechanisms ofhesperetin conjugates remain underexplored. In the present study, weaimed to characterize the sulfonation of hesperetin using humanembryonic kidney (HEK) 293 cells that stably express the SULT1A3enzyme and determine the contribution of MRP4 to the excretion ofhesperetin sulfates. SULT1A3-overexpressing HEK293 cells wereestablished by the stable transfection of a lentiviral vector carryingSULT1A3 cDNA. The reaction kinetics for the sulfonation of hesperetinwas determined using the expressed SULT1A3 enzyme and cell lysatepreparation. The role of MRP4 in the excretion of hesperetin sulfateswas evaluated through two sets of independent experiments, namely,chemical inhibition (with MK-571, a specific inhibitor of MRP familytransporters) and knockdown of MRP4 by short-hairpin RNA (shRNA).

Materials and Methods

Materials

The pMD18-T plasmid carrying the SULT1A3 cDNA clone was purchasedfrom Sino Biologic Inc. (Beijing, China). The expressed SULT1A3 enzymewas purchased from XenoTech LLC (Lenexa, KS). Anti–glyceraldehyde-3-phosphate dehydrogenase antibody was purchased from Abcam (Cambridge,MA). HEK293 cells, 293T cells, and the pLVX-mCMV-ZsGreen-PGK-Purovector (9371 base pairs) were obtained from BioWit Technologies (Shenzhen,China). Anti-BCRP (catalog number TA322704), anti-MRP1 (catalog numberTA309559), anti-MRP2 (catalog number TA313641), anti-MRP3 (catalognumber TA314800), anti-MRP4 (catalog number TA327332), and anti-MRP5(catalog number TA322563) antibodies were purchased from OriGene Technol-ogies (Rockville, MD). Anti-MRP6 (catalog number bs-17766R) antibody was

purchased from Bioss Corp. (Beijing, China). Human MRP4 membrane vesicles,39-phosphoadenosine-59-phosphosulfate, MK-571, and Ko143 were purchased fromSigma-Aldrich (St. Louis, MO). Hesperetin was purchased from Aladdin Reagents(Shanghai, China). Hesperetin-39-O-sulfate (H-39-S) and hesperetin-7-O-sulfate (H-7-S)were synthesized in our laboratory using rat liver S9 fraction as the enzyme source.All other materials (typically of analytical grade or better) were used as received.

Development of SULT1A3-Overexpressing HEK293 Cells

Cloning. Human SULT1A3 cDNA was polymerase chain reaction (PCR)-amplified from the pMD18-T-SULT1A3 plasmid through EcoRI and BamHIrestriction. The forward and reverse primers were 59-CCGGAATTCGCCAC-CATGGAGCTGATCCAGGACACCTC-39 and 59-CGCGGATCCTCACAGCT-CAGAGCGGAAGCTGAGGCT-39, respectively. The 50-ml PCR mixturecontained 10 ml of 5� FastPfu Buffer, 4 ml of 2.5 mM dNTPs (deoxynucleotidetriphosphates), 2ml of each primer (10mM), 1ml of template DNA (100 ng/ml), and1 ml FastPfu polymerase (TransGen Biotech, Beijing, China). The PCR programconsisted of an initial denaturation at 95�C for 3 minutes, 35 cycles of denaturationat 95�C for 20 seconds, annealing at 55�C for 30 seconds, extension at 72�C for2 minutes, and a final extension at 72�C for 5 minutes. The resultant PCR productswere separated by gel electrophoresis (1% agarose), and the 888–base pair fragment(corresponding to SULT1A3 cDNA) was collected and purified. The obtainedSULT1A3 cDNA was then subcloned to the pLVX-mCMV-ZsGreen-PGK-Purovector using the T4 DNA ligase (NEB, Beverly, MA). The recombinant plasmidswere transformed into Escherichia coli JM109 cells. After ampicillin selection,several cloneswere picked up and analyzed for the presence of target cDNAon a 1%agarose gel. The recombinant plasmids in positive colonies were prepared andpurified by a Plasmid Maxi preparation kit (Qiagen, Hilden, Germany). The clonedSLULT gene was sequenced within the vector construct [forward primer: CMV-F(59-CGCAAATGGGCGGTAGGCGTG-39); reverse primer: pLVX-SULT1A3-BamHI-R (59-CGCGGATCCTCACAGCTCAGAGCGGAAGCTGAGGCT-39)].

Lentiviral Vector Production. Lentiviral vectors were produced by transienttransfection of plasmid DNAs into 293T cells as described (Quan et al., 2015).

Cell Transfection. HEK293 cells were seeded at a density of 6 � 105 cells/well in a six-well plate and maintained at 37�C under 5% CO2 in Dulbecco’smodified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS).On day 2, the culture medium was changed to 100% DMEM and the lentiviruses(MOI = 10) were introduced. After a 2-hour transfection, the cells weremaintained in DMEM containing 10% FBS. On day 4, the cells were cultured inDMEM containing 10% FBS and 6 mg/ml puromycin and the medium waschanged every 2 or 3 days. After 1 week, the medium was changed to DMEMcontaining 10% FBS and 2 mg/ml puromycin. Once 100% confluence wasreached, the cells were collected and processed for DNA identification. Stablytransfected cells (named SULT293 cells) were obtained after continuous culturefor two passages. The transfection efficiency was evaluated by a fluorescencemicroscopy (Olympus IX71; Olympus Optical Co. Ltd, Tokyo, Japan).

Transient Transfection of shRNA Plasmids

The shRNAplasmids targetingMRP4have been constructed in our previous study(Quan et al., 2015). The shRNA plasmids were transiently transfected into SULT293cells as described (Quan et al., 2015). In brief, the SULT293 cells were seeded ata density of 2.0� 105 cells/well in a six-well plate and maintained at 37�C under 5%CO2 inDMEMcontaining 10%FBS.On the next day, the plasmid construct carryingthe shRNA or scramble (4 mg) was transfected into the cells using Polyfectineaccording to the manufacturer’s protocol (Biowit Technologies, Shenzhen, China).Cells were ready for excretion experiments 48 hours after transfection.

Reverse Transcription–Polymerase Chain Reaction

Cells were collected, and total RNA isolation was performed using the TRIzolextraction method. The total RNA was converted to cDNA using the iScript cDNAsynthesis kit according to themanufacturer’s protocol (Bio-Rad, Hercules, CA). ThePCR conditions were as follows: 3-minute denaturation at 95�C, followed by 30cycles of 20 seconds at 95�C, 30 seconds at 55�C, and 30 seconds at 72�C, anda final step of 72�C for 5 minutes using Taq DNA polymerase (TransGen Biotech).The primer sequences of BCRP, MRP1, MRP2, MRP3, and MRP4 can be foundin our previous publication (Quan et al., 2015). The forward and reverse primersfor MRP5 were 59-GAGAACTCGACCGTTGGAATG-39 and 59-TTCGCAGG-GAAGCAGCGTCTGG-39, respectively. The forward and reverse primers for

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MRP6 were 59-AAGATGGTGCTTGGATTCGC-39 and 59-CAGAGACAGG-CATAGGTAGGTGGA-39, respectively. After reverse transcription–polymerasechain reaction, agarose gel electrophoresis and UV visualization were used todetermine the relative amounts of PCR products.

Quantitative Real-Time PCR

Quantitative PCR (qPCR) experiments were performed using the TRIzolextraction method as described in our previous publication (Zhang et al., 2015). Inbrief, the total RNA was converted to cDNA using the iScript cDNA synthesis kit(Bio-Rad). The PCR conditions were as follows: 30-second denaturation at 95�C,followed by 45 cycles of 10 seconds at 95�C, 30 seconds at 60�C, and 30 seconds at72�C, and a final step of 1 minute at 95�C, 1 minute at 55�C, and 1 minute at 95�C.Each sample contained 0.2 mg cDNA in 10 ml SYBR green/Flourescein qPCRMaster Mix (Fermentas, Canada) and 8 pmol of each primer in a final volume of20 ml. The relative amount of each test mRNA was normalized to the level ofglyceraldehyde-3-phosphate dehydrogenase, and the data were analyzed accordingto the 2–DDCT method.

Rat Liver S9 Fraction Preparation

Liver S9 fraction was prepared using the published method (Zhu et al., 2010).Briefly, rats (n = 10) that were fasted overnight with access to water wereeuthanized. The livers were collected (4�C) and cut into tiny pieces, followed bysuspension in the homogenization buffer. Homogenization was performed witha motorized Teflon/glass homogenizer. After 15-minute centrifugation (9000g)at 4�C, the fat layer and pellet were discarded and the supernatant (i.e., S9fraction) was collected and stored at 280�C until use.

Preparation of Cell Lysate

SULT293 cells collected in 50 mM potassium phosphate buffer (pH 7.4) weredisrupted by sonication for 15 minutes in an ice-cold water bath. Cell lysate wasobtained by centrifugation (4�C) at 1000g for 5 minutes. Protein concentration wasdetermined by theBio-Rad protein assay kit using bovine serumalbumin as a standard.

Sulfonation Assay

Sulfonation activities were measured following our published procedures(Meng et al., 2012). In brief, SULT293 cell lysate or expressed SULT1A3enzyme at a final concentration of 0.1 mg protein/ml was added to 100 mM39-phosphoadenosine-59-phosphosulfate and chrysin/apigenin (at varying con-centrations) in a total reaction volume of 200 ml. The mixture was incubated at37�C for 30 minutes. The reaction was stopped by adding 100 ml of ice-coldacetonitrile. The samples were then centrifuged at 18,000g for 15minutes and thesupernatant was analyzed by ultra performance liquid chromatography (UPLC).

Sulfate Excretion Experiments

The experimental procedures for sulfate excretion were similar to those forglucuronide excretion detailed previously (Quan et al., 2015; Zhang et al., 2015).

In brief, the cells were incubated with Hank’s buffered salt solution containinghesperetin (2.5 or 10 mM) at 37�C. Transporter inhibitors, when used, werecoincubated with hesperetin. At each time point (0.5, 1, 1.5, and 2 hours),a 200-ml aliquot of incubation medium was sampled and immediately replacedwith the same volume of dosing solution. The samples were subjected to UPLCanalyses to determine the sulfate concentrations. After sampling at the last timepoint, the cells were collected and processed to measure the intracellular amountsof sulfate conjugates. The excretion rate of intracellular sulfate was calculatedexactly as described in our publications (Quan et al., 2015; Zhang et al., 2015).The apparent efflux clearance (CLef,app) was derived as the excretion rate dividedby Ci (Ci was the intracellular concentration of sulfate).

A different set of experiments was performed to obtain the hesperetin/sulfateslevels (both extracellular and intracellular) versus time profiles for pharmaco-kinetic modeling as described (Sun et al., 2015). In brief, the cells were incubatedwith hesperetin at a dose of 5 nmol. At each time point (i.e., 20, 40, 60, 80, 100,120, and 140 minutes), the incubation medium was sampled from the culturewells (n = 3). The cells were processed, and intracellular aglycone and sulfateswere measured as described above.

Vesicular Transport Assay

The vesicular transport assay was performed using the rapid filtrationtechnique as described (Wu et al., 2012). In brief, MRP4 membrane vesicleswere incubated with H-39-S or H-7-S in the presence or absence of ATP.Vesicular uptake of sulfate was terminated by the addition of an ice-coldtransport buffer, followed by rapid filtration with class F glass fiber filters (poresize: 0.45 mm). Filters were washed, cut, and transferred to a solution of 50%methanol. After sonication for 15 minutes and centrifugation at 18,000g for15 minutes, the supernatant was collected and subjected to UPLC analysis. ATP-dependent transport was calculated by subtracting the values obtained in thepresence of AMP from those in the presence of ATP.

Sulfate Quantification by UPLC Analysis

The concentrations of hesperetin sulfates were determined by the WatersACQUITY UPLC system (Milford, MA), which was equipped with an ethylenebridged hybrid column (2.1 � 50 mm, 1.7 mm). Elution was performed usinga gradient of 2.5 mM ammonium acetate in water (mobile phase A) versusacetonitrile (mobile phase B) at a flow rate of 0.4 ml/min. The gradient programwas 10% B at 0–0.5 minutes, 10 to 90% B at 0.5–2.8 minutes, 90% B at 2.8–3.3minutes, and 90 to 10%B at 3.3–4minutes. The detection wavelength was 287 nm.

Immunoblotting

Immunoblotting was performed as described (Quan et al., 2015). In brief, thecell lysate (40 mg of total protein) was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (8% acrylamide gels) and transferred topolyvinylidene fluoride membranes (Millipore, Bedford, MA). Blots were probedwith the transporter antibodies (i.e., anti-BCRP, anti-MRP1, anti-MRP2, anti-MRP3,

Fig. 1. Schematic representation of a two-compartment model that depicts thesulfonation of hesperetin and excretion of its sulfate metabolites in SULT293 cells.Please refer to the text for the definition of each parameter. ET, efflux transporter; H,hesperetin; S1, H-39-S; S2, H-7-S. The subscripts m and c denote the extracellularand cellular compartments, respectively.

Fig. 2. Representative UPLC chromatograms showing that SULT293 cells wereactive in the generation and excretion of hesperetin sulfates. The UPLC samples weregenerated after incubation of the cells with hesperetin (10 mM) at different time points.

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anti-MRP4, anti-MRP5, and anti-MRP6) at a dilution of 1:1000, followedby horseradish peroxidase–conjugated rabbit anti-goat IgG (Santa Cruz Bio-technology, Santa Cruz, CA). Protein bands were visualized by enhancedchemiluminescence.

Modeling of Enzyme Kinetics

Kinetic parameters were derived by the fitting substrate inhibition equation (eq.1) to the data of the reaction rates versus substrate concentrations. Substrateinhibition refers to the inhibition of enzyme activity at high substrate concentrations.Parameter estimation was performed using Graphpad Prism 5 (San Diego, CA).

V ¼ Vmax × ½S�Km þ ½S�

�1þ ½S�

Ksi

� ð1Þ

where Km is the Michaelis constant, Vmax is the maximal velocity, and Ksi is thesubstrate inhibition constant.

Pharmacokinetic Modeling and Data Fitting

A two-compartment model (Fig. 1), consisting of extracellular and cellularcompartments, was established to describe the transport, metabolism, andexcretion processes in SULT293 cells. The mass balance equations forthe model are shown below (eqs. 2–7). The subscripts m and c denoted theextracellular and cellular compartments, respectively. Transport of hesperetin(H) across the cell membrane was controlled by the passive diffusion mechanism(represented by the transport clearance CLd). Formation of H-39-S (S1) and H-7-S(S2) by SULT1A3 obeyed the substrate inhibition kinetics (Vmax,1, Km,1, andKsi,1 for S1; Vmax,2, Km,2, and Ksi,2 for S2). Sulfate excretion was a saturableprocess described by Jmax and K9m (Jmax,1 and K9m,1 for S1; Jmax,2 and K9m,2 for

S2). fu denoted the unbound fraction of hesperetin in the cell compartment. Themodel assumed that binding of sulfates (polar compounds) to cellular proteinswas negligible.

Model construction and data fitting were performed using MATLAB(Mathsworks Inc., Natick, MA). In data fitting, the Km and Ksi values werefixed as the corresponding values derived from the in vitro sulfonation assay.Also, the K9m values were fixed as the corresponding values derived from the invitro transport kinetics with MRP4 membrane vesicles.

dHm

dt¼ 2

CLdVm

Hm þ CLdVc

fuHc ð2Þ

dHc

dt¼ CLd

VmHm 2

CLdVc

fuHc 2fuHcVmax;1

VcKm;1 þ fuHc þ ð fuHcÞ2VcKsi;1

2fuHcVmax;2


ð3Þ

dS1mdt

¼ S1cJmax;1

VcK9m;1 þ S1c

ð4Þ

dS2mdt

¼ S2cJmax;2

VcK9m;2 þ S2c

ð5Þ

dS1cdt

¼ fuHcVmax;1


2S1cJmax;1

VcK9m;1 þ S1c

ð6Þ

dS2cdt

¼ fuHcVmax;2


2S2cJmax;2

VcK9m;2 þ S2c

ð7Þ

Fig. 3. Disposition of hesperetin in SULT293 cells at different doses. (A) Excretionrates of hesperetin sulfates at different loading doses. (B) Intracellular amounts ofhesperetin and its sulfates at 2 hours under different loading doses. ***P , 0.001.Each data point was the average of three determinations, with the error barrepresenting the standard deviation (n = 3).

Fig. 4. Kinetic profiles for regiospecific sulfonation of hesperetin by SULT293 celllysate (A) and expressed SULT1A3 enzyme (B). Each data point was the average ofthree determinations, with the error bar representing the standard deviation (n = 3).


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Statistical Analysis

Data are expressed as mean 6 S.D. Mean differences between treatment andcontrol groups were analyzed by Student’s t test usingGraphpad Prism 5 (SanDiego,CA). The level of significance was set at *P, 0.05, **P, 0.01, or ***P, 0.001.

Results

Generation and Excretion of Hesperetin Sulfates in SULT293Cell Model. No metabolites were found in the medium or within the

cells after incubation of wild-type HEK293 cells with hesperetin. Incontrast, two monosulfates (i.e., H-39-S and H-7-S) were generatedfrom hesperetin by SULT293 cells (Fig. 2). Further, the excretedamounts of sulfate metabolites increased with the incubation time (Fig.2). This enabled us to derive the rate of sulfate excretion at differentdoses of hesperetin. The rates of sulfate excretion significantlyincreased (P , 0.001) as the dose was increased from 2.5 to 10 mM(Fig. 3A). Increased sulfate excretion was associated with an elevatedlevel of hesperetin within the cells (Fig. 3B). The detection of sulfates

TABLE 1

Kinetic parameters derived for hesperetin sulfonation by cell lysate preparation and expressed SULT1A3 enzyme

Data are represented by mean 6 S.E.

Enzyme Source Metabolite Vmax Km Ksi CLin Fitted Model

nmol/min per mg mM mM ml/min per mg

Cell lysateH-39-S 0.66 6 0.08 12.9 6 2.41 58.1 6 13.7 51.2 6 6.13 SIH-7-S 0.29 6 0.04 14.8 6 2.87 49.1 6 11.6 19.6 6 3.56 SI

SULT1A3H-39-S 3.46 6 0.45 18.5 6 3.44 37.9 6 8.17 187 6 10.2 SIH-7-S 1.43 6 0.15 18.4 6 2.82 36.4 6 6.42 77.6 6 8.33 SI

SI, substrate inhibition model.

Fig. 5. Expression of efflux transporters in wild-type (WT) and transfected (SULT293) HEK293 cells. (A) mRNA expression of BCRP and six MRP family transporters inHEK293 and SULT293 cells detected by reverse transcription–polymerase chain reaction. (B) qPCR measurements of BCRP, MRP1, MRP4, and MRP5 in SULT293 cells.(C) Protein expression of BCRP and six MRP family transporters in HEK293 and SULT293 cells.

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(10–39 pmol/mg protein) inside the cells was additional evidencethat the SULT293 cells were capable of catalyzing sulfonation reac-tions (Fig. 3B). The results overall indicated that overexpression ofSULT1A3 generated a cell line that was metabolically active only at thesulfonation pathway. Thus, the SULT293 cells were a useful model toinvestigate the transport and metabolism of drugs and their sulfatemetabolites.Sulfonation Kinetics of Hesperetin with SULT293 Cell Lysate

and Expressed SULT1A3 Enzyme. Both SULT293 cell lysate andrecombinant SULT1A3 generated two sulfate metabolites (i.e., H-39-Sand H-7-S) from hesperetin. H-39-S formation mediated by cell lysate

followed the substrate inhibition kinetics (Vmax = 0.66 nmol/min permg; Km = 12.9 mM; and Ksi = 58.1 mM) (Fig. 4A; Table 1). Theformation kinetics of H-7-S were also well described by the substrateinhibition model (Vmax = 0.29 nmol/min per mg; Km = 14.8 mM; andKsi = 49.1 mM) (Fig. 4A; Table 1). Likewise, regiospecific sulfonationof hesperetin by recombinant SULT1A3 obeyed the substrate inhibitionkinetics (Vmax = 3.46 nmol/min per mg, Km = 18.5 mM, and Ksi = 37.9 mMforH-39-S;Vmax = 1.43nmol/min permg,Km=18.4mM,andKsi = 36.4mMfor H-7-S) (Fig. 4B; Table 1).The Km and Ksi values of regiospecific sulfonation derived from the

cell lysate were similar (P . 0.05) to their corresponding values from

Fig. 6. Effects of MK-571 on sulfonationactivity. (A) Effects of MK-571 on sulfonationof hesperetin (2.5 mM) mediated by SULT293cell lysate. (B) Effects of MK-571 on sulfona-tion of hesperetin (10 mM) mediated bySULT293 cell lysate. (C) Effects of MK-571on sulfonation of hesperetin (2.5 mM) mediatedby the expressed SULT1A3 enzyme. (D) Effectsof MK-571 on sulfonation of hesperetin (10 mM)mediated by the expressed SULT1A3 enzyme.Each data point was the average of threedeterminations, with the error bar representingthe standard deviation (n = 3).

Fig. 7. Effects of MK-571 on sulfate disposition afterincubation of SULT293 cells with hesperetin (2.5 mM).(A) Effects of MK-571 on the excretion rates ofhesperetin sulfates. (B) Effects of MK-571 on theintracellular levels of hesperetin sulfates. (C) Effects ofMK-571 on the efflux clearances (CLef,app) of hesperetinsulfates. Each data point was the average of threedeterminations, with the error bar representing thestandard deviation (n = 3). H-39-S, hesperetin-39-O-sulfate; H-7-S, hesperetin-7-O-sulfate. ***P , 0.001compared with vehicle control.


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the recombinant enzyme (Table 1). This indicated that the sulfonationactivity of the cell lysate arose from the SULT1A3 enzyme stablyexpressed in the cells. However, the Vmax values (closely related to theenzyme concentration) derived from the SULT1A3 enzymewere nearly5 times those from the SULT293 cell lysate (Table 1). This was notunexpected because the SULT1A3 enzyme was much more concen-trated in the recombinant material compared with the cell lysatepreparation. Furthermore, both the cell lysate and SULT1A3 enzymeshowed a conjugation preference for the 39-OH group over the 7-OHgroup (Fig. 4). The intrinsic clearance values (CLint, reflective ofcatalytic efficiency) of 39-O-sulfonation were much larger (P, 0.001)than those of 7-O-sulfonation (Table 1).Expression of Efflux Transporters in HEK293 and SULT293

Cells. Cellular expression of BCRP and MRP family proteins was

measured at both mRNA and protein levels. HEK293 cells expressedthe mRNAs of BCRP, MRP1, MRP4, and MRP5 according to thereverse transcription–polymerase chain reaction (Fig. 5A). Also, theqPCR results showed that the mRNA levels of BCRP and MRP4 wereover 10 times those of MRP1 andMRP5 (Fig. 5B). Further, only BCRPand MRP4 proteins were detected in the cells by western blotting (Fig.5C). The results suggested that the two exporters, BCRP and MRP4,with dominant expression were potential contributors to the excretionof sulfate metabolites. It was noteworthy that the engineered SULT293cells showed an identical expression of the transporters (Fig. 5),indicating that transfection of SULT1A3 did not alter transporterexpression.Effects of MK-571 on Hesperetin Sulfonation. The effects of MK-

571, a pan-MRP inhibitor, on hesperetin sulfonation were determined

Fig. 8. Effects of MK-571 on sulfate disposition afterincubation of SULT293 cells with hesperetin (10 mM).(A) Effects of MK-571 on the excretion rates ofhesperetin sulfates. (B) Effects of MK-571 on theintracellular levels of hesperetin sulfates. (C) Effects ofMK-571 on the efflux clearances (CLef,app) of hesperetinsulfates. Each data point was the average of threedeterminations, with the error bar representing thestandard deviation (n = 3). ***P , 0.001 comparedwith vehicle control.

Fig. 9. Effects of MRP4 silencing on sulfate disposi-tion. (A) Effects of MRP4 silencing on the excretionrates of hesperetin sulfates. (B) Effects of MRP4silencing on the intracellular levels of hesperetinsulfates. (C) Effects of MRP4 silencing on the effluxclearances (CLef,app) of hesperetin sulfates. Each datapoint was the average of three determinations, with theerror bar representing the standard deviation (n = 3).Statistically significant differences between scrambleand shRNA-treated cells are indicated by asterisks(*P , 0.05; **P , 0.01; and ***P , 0.001).

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using the SULT293 cell lysate and the expressed SULT1A3 enzyme.MK-571 at all tested concentrations (5–20 mM) did not show anymodulatory effects on hesperetin sulfonation mediated by cell lysate(Fig. 6, A and B). Neither H-39-S nor H-7-S formation was altered in thepresence of MK-571 (Fig. 6, A and B). Likewise, sulfonation ofhesperetin by expressed SULT1A3 was not modulated by MK-571(Fig. 6, C and D). The results indicated that the MRP inhibitor MK-571had no effects on SULT1A3 activity.Effects of MK-571 on Excretion of Hesperetin Sulfates. At a low

loading dose (2.5 mM) of hesperetin, MK-571 caused substantialreductions (.57.5%; P , 0.001) in sulfate excretion (Fig. 7A). It alsoled to marked elevations (.256%; P , 0.001) in sulfate accumulation(i.e., intracellular sulfates) (Fig. 7B). Then, it was not surprising that theapparent efflux clearances (CLef,app) of sulfates were dramaticallysuppressed (,10.4% of control) in the presence ofMK-571 (Fig. 7C). Itwas noteworthy that MK-571 at 20 mM essentially abolished cellularexcretion of both H-39-S and H-7-S as the sulfate excretion was only 6%of the control (Fig. 7A).Similar effects of MK-571 on sulfate disposition were observed when

a higher loading dose (10 mM) of hesperetin was used (Fig. 8). MK-571(5–20 mM) markedly decreased the excretion rates (.84.4%; P, 0.001)of hesperetin sulfates while increasing (.173%) their intracellular levels(Fig. 8, A and B). Accordingly, the CLef,app values of sulfates weresubstantially decreased (.91.8%; P, 0.001) (Fig. 8C). It was clear thatthe extent of excretion inhibition depended on the inhibitor concentration.A higher extent of excretion inhibition was observed with a higherinhibitor concentration (Figs. 7 and 8). Taken together, our results stronglysuggested that the MRP family transporter, more specifically, MRP4 (theonly MRP protein expressed in the cells), was an important contributor tothe excretion of hesperetin sulfates.Effects of MRP4 Knockdown on Sulfate Excretion. MRP4 was

knocked down by transient transfection of shRNA. The selectedshRNA was shown to significantly decrease the expression of targettransporter MRP4 by;65% in our previous studies (Quan et al., 2015;Zhang et al., 2015). MRP4 knockdown led to substantial reductions(.47.1%; P , 0.01) in the rates of sulfate excretion (Fig. 9A). Onthe contrary, knockdown of MRP4 caused significant elevations(.170%; P , 0.05) in the intracellular levels of sulfates (Fig. 9B).As a consequence, the CLef,app values of sulfates were reduced to 23.9–35.3% of the control (P , 0.001) (Fig. 9C). The marked changes insulfate excretion caused by decreasing MRP4 expression was addi-tional evidence that MRP4 played a critical role in the excretion ofhesperetin sulfates.Transport Kinetics of Hesperetin Sulfates with Human

MRP4. Transport of hesperetin sulfates (H-39-S and H-7-S) by humanMRP4 was investigated using membrane vesicles (Fig. 10). It was clearthat hesperetin sulfates were good substrates for transport by human MRP4(Fig. 10). Transport of bothH-39-S andH-7-S byMRP4 followedMichaelis-Menten kinetics (Jmax = 18.4 pmol/min per mg and K9m = 2.74 mM forH-39-S; Jmax = 15.5 pmol/min per mg and K9m = 3.24 mM for H-7-S)(Fig. 10). The Jmax andK9m values of H-39-S were similar (P. 0.05) to thecorresponding values of H-7-S, indicating that MRP4 had an equaltransport activity toward the positional sulfate isomers (Fig. 10).Effects of Ko143 on Excretion of Hesperetin Sulfates. Ko143 is

a potent and selective inhibitor ofBCRP.However, use ofKo143 (5–20mM)did not cause any changes in either sulfate excretion or sulfateaccumulation (Fig. 11). We also found that Ko143 did not alter thesulfonation of hesperetin by SULT1A3 (data not shown). The resultsindicated that the role of BCRP in the excretion of hesperetin sulfates wasnone or negligible, although BCRP was expressed in the cells (Fig. 5).Mechanistic Pharmacokinetic Model for Hesperetin/Sulfate

Disposition in SULT293 Cells. The concentration-time profiles were

determined for extracellular hesperetin/sulfates and intracellularhesperetin/sulfates at a dose of 5 nmol hesperetin (Fig. 12). A two-compartment pharmacokineticmodel (Fig. 1) integrating drug uptake andsulfonation with sulfate excretion was used to describe the data (Fig. 12;Table 2). Mechanistic fitting was performed by fixing several parameters(independent of the model system) to those values derived from in vitrocharacterization (i.e.,Km andKsi from the sulfonation assay andK9m fromthe MRP4 vesicular transport assay) (Table 2). The coefficients ofvariations for fitted parameters were ,20%, which is suggestive ofadequate fitting (Table 2). Adequate fitting of the model to data helped usto fully understand the cellular deposition processes of hesperetin via thesulfonation pathway (Fig. 12). Following rapid uptake into the cells bypassive diffusion, hesperetin was conjugated to form two sulfate isomers(Fig. 13). The generated sulfates were then excreted into the extracellularcompartment primary via the action of MRP4 (Fig. 13).

Discussion

In this study, we characterized the sulfonation of hesperetin andexcretion of its sulfate metabolites using HEK293 cells overexpressingSULT1A3 (named SULT293 cells). The SULT293 cells were able tosulfate hesperetin at both the 39-OH and 7-OH groups, owing to stableexpression of the SULT1A3 enzyme. The generation and excretion oftwo sulfates (H-39-S and H-7-S) enabled us to simultaneously evaluatethe excretion of positional isomers of hesperetin sulfates. It was foundfor the first time that excretion of both sulfate isomers in the cells wasprimarily contributed by MRP4. The evidence was strong and includedfour sets of independent results. First, inhibition of sulfate excretion by

Fig. 10. Kinetic profiles for transport of H-39-S (A) and H-7-S (B) with MRP4membrane vesicles. The units for K9m and Jmax are mM and pmol/min per mgprotein, respectively. Each data point was the average of three determinations, withthe error bar representing the standard deviation (n = 3).


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MK-571 (a selective inhibitor of MRP family proteins) was essentiallycomplete (;94% inhibition at 20mMMK-571) (Figs. 7 and 8). Second,knockdown of MRP4 led to reduced excretion of hesperetin sulfates(Fig. 9). Third, hesperetin sulfates were high-affinity substrates fortransport by MRP4 according to the vesicular transport assay (Fig. 10).Fourth, the mechanistic pharmacokinetic model, assumingMRP4 as theonly sulfate exporter (i.e., the model parameters K9m,1 and K9m,2 valueswere fixed at those values derived from MRP4 membrane vesiclesduring model fitting), was well fitted to the experimental data (Fig. 12).Elucidating the transport mechanisms for phase II conjugates

(sulfates and glucuronides) of hesperidin assumes great importancebecause 1) these hydrophilic conjugates are the main circulatingmetabolites; and 2) the conjugates possess many types of biologicactivities, such as antioxidant and anti-inflammatory effects (Proteggenteet al., 2003; Trzeciakiewicz et al., 2010; Yang et al., 2012; Yamamotoet al., 2013; Gamo et al., 2014). It has been shown that multipletransporters (BCRP, MRP2, and MRP3) are potentially responsible forefflux transport of hesperetin glucuronides (Brand et al., 2008, 2011).Our work was the first report that excretion of hesperetin sulfateswas mainly mediated by MRP4 in HEK293 cells. Due to significantexpression in the liver and kidney (Ritter et al., 2005; Russel et al.,2008), MRP4 should be an important contributor to hepatic/renalexcretion of hesperetin sulfates. Our finding that MRP4 transportedsulfate metabolites was consistent with a previous study, in which MRP4participated in hepatic excretion of the sulfate metabolites of acetamin-ophen, 4-methylumbelliferone, and harmol (Zamek-Gliszczynski et al.,2006a). However, edaravone sulfate was not transported by MRP4 in thestudy of Mizuno et al. (2007). Therefore, substrate recognition of sulfate

conjugates by MRP4 was not solely dependent on the sulfonate group.The aglycone part also played an important role.Although HEK293 cells expressed the BCRP protein (Fig. 5),

contribution of BCRP to the excretion of hesperetin sulfates was noneor negligible. This was because Ko143 (a potent and selective inhibitor ofBCRP) did not alter the sulfate excretion at all (Fig. 11) (Allen et al.,2002). In fact, the use of shRNA targeting BCRP also did not changesulfate disposition (Quan et al., 2015; Zhang et al., 2015). Hence, wewereconvinced that BCRP was not involved in the efflux transport ofhesperetin sulfates. However, this finding appeared to be inconsistentwith a previous study of Brand et al. (2008), in which BCRP played a rolein the apical efflux of H-7-S in Caco-2 cells. Although data interpretationby Brand et al. (2008) may be confounded by the fact that Ko143 has thepotential to alter the activities of conjugating enzymes (Quan et al., 2015),additional investigations were needed to address this discrepancy.The finding that SULT1A3 catalyzed the conjugation of hesperetin at

both the 39-OH and 7-OH groups, with a positional preference for theformer, was consistent with the study of Brand et al. (2010a). In thecurrent work, regiospecific sulfonation of hesperetin by the SULT1A3and SULT293 cell lysate followed substrate inhibition kinetics(i.e., inhibition of enzyme activity at high substrate concentrations)(Fig. 4; Table 1). This agreed well with a previous study by Huang et al.(2009), in which SULT1A3-mediated sulfonation of hesperetin alsoshowed substrate inhibition kinetics. However, Brand et al. (2010a)reported that regiospecific sulfonation of hesperetin by SULT1A3obeyed Michaelis-Menten kinetics. It remained to be clarified whySULT1A3 behaved differently in the Brand et al. study. Nevertheless, itshould be noted that sulfonation of many chemicals (e.g., dopamine,catechin, and eriodictyol) by SULT1A3 displayed the substrate in-hibition phenomenon (Wu, 2011).Thewild-typeHEK293 cellswere unable to conjugate hesperetin due to

a lack of expression of SULT1A3. However, HEK293 cells were reportedto intrinsically express other types of SULT enzymes, such as SULT1C1and SULT1E1 (Kapoor et al., 2007; Sheng and Acquaah-Mensah, 2011).

Fig. 12. Pharmacokinetic modeling of hesperetin disposition in SULT293 cellsat a dose of 5 nmol. (A) Extracellular hesperetin level versus time profile. (B)Extracellular sulfate levels versus time profiles. (C) Intracellular hesperetin levelversus time profile. (D) Intracellular sulfate levels versus time profiles. Eachdata point was the average of three determinations, with the error bar representingthe standard deviation (n = 3). Solid lines are the predicted data from thepharmacokinetic model.

Fig. 11. Effects of Ko143 on sulfate disposition after incubation of SULT293 cellswith hesperetin (10 mM). (A) Effects of Ko143 on the excretion rates of hesperetinsulfates. (B) Effects of Ko143 on the intracellular levels of hesperetin sulfates. Eachdata point was the average of three determinations, with the error bar representingthe standard deviation (n = 3).

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Further, the SULT1E1 enzyme shows in vitro conjugation activity towardhesperetin, although the relative activities between SULT1E1 andSULT1A3were not determined (as the enzyme levels in the recombinantmaterials were unknown) (Brand et al., 2010a). The exact reasonwas unknown as to why wild-type HEK293 cells were inactive inconjugating hesperetin. It was hypothesized that the inability of HEK293cells tometabolize hesperetin was due to a low expression of SULT1E1 inHEK293 cells and/or low sulfonation activity of SULT1E1 toward thecompound.The effects of the transport inhibitor MK-571 on SULT1A3-mediated

sulfonation of hesperetin were determined using both the cell lysatepreparation and recombinant enzyme (Fig. 6). The information wasnecessary to accurately interpret the results of sulfate excretion inhibition.This was because modulation of enzyme activity by the transporterinhibitors was a confounding factor to identification of the transportersusing the chemical inhibition method (Quan et al., 2015). It was foundthat MK-571 did not alter the sulfonation rates of hesperetin. Therefore,changes in the excretion profiles of hesperetin sulfates caused byMK-571were solely ascribed to the suppression of MRP4 activity.The role of MRP4 in the excretion of hesperetin sulfates was

confirmed through determination of the effects ofMRP4 knockdown onsulfate excretion (Fig. 9). The efficiency of protein knockdown highlydepended on the shRNA fragments. We have designed four differentshRNA sequences for MRP4 and evaluated their performance inknocking down MRP4 in our previous study (Zhang et al., 2015).The best-performing shRNA was obtained based on the interferenceefficiency and was used to silence MRP4 in the present study. Theselected shRNA caused a significant reduction (;65%) in the targetprotein MRP4 while showing no effects on the off-target transporters(Zhang et al., 2015).Sulfonation of hesperetin in vitro favored the formation of H-39-S

over H-7-S (Fig. 4). The formation ratio was about 2.0 at hesperetinconcentrations of 2.5 and 10 mM. It was interesting to note that theexcretion ratio of H-39-S over H-7-S in SULT293 cells was close to 2.0at loading doses of 2.5 and 10mMhesperetin. An unchanged ratio in thesulfate isomer excretion suggested that the efflux transporter MRP4 hadsimilar transport activities toward H-39-S and H-7-S. This was supportedby vesicular transport assays that showed H-39-S and H-7-S were equallytransported by MRP4, with similar kinetic parameters (P . 0.05).However, it was not always true that the efflux transporters have equalaffinities for positional isomers of hesperetin conjugates. For instance,interaction of BCRP with hesperetin glucuronides depended on theposition of substitution. Hesperetin 7-O-glucuronide was a high-affinity

substrate of BCRP, whereas hesperetin 39-O-glucuronide was a non-substrate or poor substrate of BCRP (Brand et al., 2011).Successful establishment of an integrated pharmacokinetic model

allowed us to fully understand the cellular disposition processes ofhesperetin and its sulfate metabolites (Fig. 1). Following rapid uptakeinto the SULT293 cells by passive diffusion, hesperetin was conjugatedby SULT1A3 to form two sulfate isomers (Fig. 13). Passive transport ofhesperetin across membranes has also been documented in the literature(Brand et al., 2008). The generated sulfates were then excreted into theextracellular compartment by efflux transporter(s), with a dominantcontribution from MRP4 (Fig. 13). MRP4 appeared to be a “molecularswitch” that controlled cellular efflux of hesperetin sulfates. Thus,inhibition of MRP4 activity or decreasing MRP4 expression led toa reduced sulfate excretion (Figs. 7–9). The results highlighted that theefflux transporter MRP4 played an important role in the disposition ofsulfate metabolites (and possibly the parent compound hesperetin).The present study suggested that the SULT293 cells were an excellent

tool to investigate SULT1A3-mediated sulfonation and characterizeMRP4-mediated transport of sulfate metabolites (including positionalisomers). The SULT293 cells have an advantage over other methods/tools (such as membrane vesicles and monolayer cells overexpressinga transporter) because drug sulfates (usually lacking in commercialavailability) were not required for experimentation as the metabolitesare generated from the dosed drug by the cells. In addition, the SULT293cells were free of the concerns raised in transporter identification studiesusing membrane vesicle or monolayer cells (Fahrmayr et al., 2012).First, drug sulfates poorly cross cellular membranes by passivediffusion. Use of polarized monolayers (expressing a transporter) withadministration of the sulfate can be problematic because the sulfate maynot enter the cells (Fahrmayr et al., 2012). Second, studies with inside-outvesicles are time consuming and challenging (Fahrmayr et al., 2012).In summary, two monosulfate metabolites, H-39-S and H-7-S, were

generated from hesperetin in SULT293 cells and efficiently excretedinto the extracellular compartment. The pan-MRP inhibitor MK-571at 20 mM essentially abolished cellular excretion of both H-39-S andH-7-S, whereas Ko143 had no effects on sulfate excretion. Knockdownof MRP4 led to a substantial reduction (.47.1%) in sulfate excretion.Further, H-39-S and H-7-S were good substrates for transport by MRP4according to vesicular transport assay. Taken together, we concludedthat MRP4 dominated the excretion of hesperetin sulfates in SULT293

Fig. 13. A summary of disposition processes of hesperetin and its sulfatemetabolites in SULT293 cells.

TABLE 2

Fitted parameters for sulfonation of hesperetin and efflux of its sulfate metabolites inSULT1A3-overexpressing HEK293 cells

PK Parameters Values CV

%

CLd (ml/h) 49.9 6 9.96 19.9Km,1 (mM) 12.9a NAKsi,1 (mM) 58.1a NAVmax,1 (pmol/min) 100 6 5.17 5.2Km,2 (mM) 14.8a NAKsi,2 (mM) 49.1a NAVmax,2 (pmol/min) 49.3 6 2.75 5.6fu 0.022 6 0.001 4.5K9m,1 (mM) 2.7b NAJmax,1 (pmol/min) 29.0 6 1.41 4.9K9m,2 (mM) 3.2b NAJmax,2 (pmol/min) 25.0 6 1.68 6.7

aAssigned values from in vitro metabolism kinetics with cell lysate.bAssigned values from in vitro transport kinetics with membrane vesicles.NA, not applicable.


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cells. Due to significant expression of MRP4 in various organs/tissues,MRP4 should be a determining factor for the elimination and bodydistribution of hesperetin sulfates.

Authorship ContributionsParticipated in research design: Sun, Wang, Zhou, Lu, Wu.Conducted experiments: Sun, Zhou, Lu.Contributed new reagents or analytic tools: Wang, Ma.Performed data analysis: Sun, Wang, Zhou, Lu, Ma, Wu.Wrote or contributed to the writing of the manuscript: Sun, Wang, Wu.

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Address correspondence to: Dr. Baojian Wu, Division of Pharmaceutics, Collegeof Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632,China. E-mail: [email protected]

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