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Multidrug Resistance-Associated Protein 4 (MRP4/ABCC4) Controls Efflux Transport of Hesperetin Sulfates in Sulfotransferase 1A3–Overexpressing Human Embryonic Kidney 293 Cells

Hua Sun, Xiao Wang, Xiaotong Zhou, Danyi Lu, Zhiguo Ma and Baojian Wu
Drug Metabolism and Disposition October 2015, 43 (10) 1430-1440; DOI: https://doi.org/10.1124/dmd.115.065953

Abstract

Sulfonation is an important metabolic pathway for hesperetin. However, the mechanisms for the cellular disposition of hesperetin and its sulfate metabolites are not fully established. In this study, disposition of hesperetin via the sulfonation pathway was investigated using human embryonic kidney (HEK) 293 cells overexpressing sulfotransferase 1A3. Two monosulfates, hesperetin-3′-O-sulfate (H-3′-S) and hesperetin-7-O-sulfate (H-7-S), were rapidly generated and excreted into the extracellular compartment upon incubation of the cells with hesperetin. Regiospecific sulfonation of hesperetin by the cell lysate followed the substrate inhibition kinetics (Vmax = 0.66 nmol/min per mg, Km = 12.9 μM, and Ksi= 58.1 μM for H-3′-S; Vmax = 0.29 nmol/min per mg, Km = 14.8 μM, and Ksi= 49.1 μM for H-7-S). The pan–multidrug resistance-associated protein (MRP) inhibitor MK-571 at 20 μM essentially abolished cellular excretion of both H-3′-S and H-7-S (the excretion activities were only 6% of the control), whereas the breast cancer resistance protein–selective inhibitor Ko143 had no effects on sulfate excretion. In addition, knockdown of MRP4 led to a substantial reduction (>47.1%; P < 0.01) in sulfate excretion. Further, H-3′-S and H-7-S were good substrates for transport by MRP4 according to the vesicular transport assay. Moreover, sulfonation of hesperetin and excretion of its metabolites were well characterized by a two-compartment pharmacokinetic model that integrated drug uptake and sulfonation with MRP4-mediated sulfate excretion. In conclusion, the exporter MRP4 controlled efflux transport of hesperetin sulfates in HEK293 cells. Due to significant expression in various organs/tissues (including the liver and kidney), MRP4 should be a determining factor for the elimination and body distribution of hesperetin sulfates.

Introduction

Phase II metabolism refers to various conjugation reactions, wherein a polar moiety (e.g., sulfonate and glucuronic acid) is conjugated to the substrates. In general, phase II metabolism increases water solubility of the parent drug, thereby facilitating drug inactivation and elimination. Since more and more new drug entities are metabolized directly by phase II enzymes, phase II metabolism is becoming increasingly important in drug discovery and development (Rowland et al., 2013). Cytosolic sulfotransferases (SULTs) are a family of enzymes that catalyze the sulfonation (or sulfation) reaction (i.e., addition of a sulfonate group to the substrates), a type of phase II reaction (Klaassen and Boles, 1997). The sulfonation reaction represents an important mechanism in activity regulation 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 of SULT1 and SULT2 families, with abundant expression in the liver and intestine, play a dominant role in catalyzing sulfonation reactions (Allali-Hassani et al., 2007; Teubner et al., 2007; Riches et al., 2009).

Hesperetin (4′-methoxy-3′,5,7-trihydroxyflavanone) is a main and widespread citrus flavonoid that shows versatile health benefits, such as chemopreventive, cardioprotective, and neuroprotective effects (Manthey et al., 2001; Benavente-García and Castillo, 2008; Hwang and Yen, 2008; Hwang et al., 2012). The beneficial effects of hesperetin on health are mainly attributed to its antioxidant, anti-inflammatory, and signaling properties (Hwang et al., 2012; Roohbakhsh et al., 2015). Although showing favorable membrane permeability, hesperetin is concerned with limited oral bioavailability (Kanaze et al., 2007; Kobayashi et al., 2008). One of the main causes for the poor absorption of hesperetin is extensive phase II metabolism (i.e., glucuronidation and sulfonation) in the intestine and liver (Silberberg et al., 2006; Takumi et al., 2012). Hence, inhibition of metabolism appears to be an effective strategy 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 multiple sulfotransferase (SULT) enzymes (including SULT1A1, SULT1A3, and SULT2A1) are actively involved in conjugating hesperetin (Brand et al., 2010a). In addition, hesperetin conjugates are found to be the main circulating metabolites in humans and rodents, confirming the importance of conjugative metabolism in hesperetin disposition (Matsumoto et al., 2004; Takumi et al., 2012; Yamamoto et al., 2013). Further, the hesperetin conjugates (e.g., hesperetin-7-O-glucuronide) most likely retain the biologic activities of the parent compound (Yamamoto et al., 2013). Hence, it is essential to elucidate the disposition mechanisms for these hydrophilic metabolites.

Transport of generated sulfates out of cells is an essential step in compound elimination via the sulfonation pathway. Due to the polar nature, the sulfate conjugates cannot be transported by the passive diffusion mechanism. Transcellular transport of sulfate conjugates is facilitated by membrane transporters (Zamek-Gliszczynski et al., 2006a,b, 2011). Investigations on the disposition of several drugs (i.e., acetaminophen, 4-methylumbelliferone, and ethinylestradiol) in rodents have revealed that the efflux transporters breast cancer resistance 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 Brand et al. (2008) has shown that BCRP is involved in apical excretion of hesperetin conjugates in Caco-2 cells. However, there is no information regarding 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 of numerous xenobiotics/drugs, including antiviral, cardiovascular, and antibiotic drugs (Russel et al., 2008). Hence, like other transporters, MRP4 plays an important role in the elimination and body distribution of drugs (Giacomini et al., 2010; DeGorter et al., 2012). Furthermore, MRP4 is involved in cellular communication and signaling because of its marked ability to transport many signaling molecules, such as cyclic nucleotides, eicosanoids, and urates (Russel et al., 2008).

Phase II conjugates are the main circulating and potentially active metabolites of hesperetin. However, the disposition mechanisms of hesperetin conjugates remain underexplored. In the present study, we aimed to characterize the sulfonation of hesperetin using human embryonic kidney (HEK) 293 cells that stably express the SULT1A3 enzyme and determine the contribution of MRP4 to the excretion of hesperetin sulfates. SULT1A3-overexpressing HEK293 cells were established by the stable transfection of a lentiviral vector carrying SULT1A3 cDNA. The reaction kinetics for the sulfonation of hesperetin was determined using the expressed SULT1A3 enzyme and cell lysate preparation. The role of MRP4 in the excretion of hesperetin sulfates was evaluated through two sets of independent experiments, namely, chemical inhibition (with MK-571, a specific inhibitor of MRP family transporters) and knockdown of MRP4 by short-hairpin RNA (shRNA).

Materials and Methods

Materials

The pMD18-T plasmid carrying the SULT1A3 cDNA clone was purchased from Sino Biologic Inc. (Beijing, China). The expressed SULT1A3 enzyme was 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-Puro vector (9371 base pairs) were obtained from BioWit Technologies (Shenzhen, China). Anti-BCRP (catalog number TA322704), anti-MRP1 (catalog number TA309559), anti-MRP2 (catalog number TA313641), anti-MRP3 (catalog number TA314800), anti-MRP4 (catalog number TA327332), and anti-MRP5 (catalog number TA322563) antibodies were purchased from OriGene Technologies (Rockville, MD). Anti-MRP6 (catalog number bs-17766R) antibody was purchased from Bioss Corp. (Beijing, China). Human MRP4 membrane vesicles, 3′-phosphoadenosine-5′-phosphosulfate, MK-571, and Ko143 were purchased from Sigma-Aldrich (St. Louis, MO). Hesperetin was purchased from Aladdin Reagents (Shanghai, China). Hesperetin-3′-O-sulfate (H-3′-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 BamHI restriction. The forward and reverse primers were 5′-CCGGAATTCGCCACCATGGAGCTGATCCAGGACACCTC-3′ and 5′-CGCGGATCCTCACAGCTCAGAGCGGAAGCTGAGGCT-3′, respectively. The 50-μl PCR mixture contained 10 μl of 5× FastPfu Buffer, 4 μl of 2.5 mM dNTPs (deoxynucleotide triphosphates), 2 μl of each primer (10 μM), 1 μl of template DNA (100 ng/μl), and 1 μl FastPfu polymerase (TransGen Biotech, Beijing, China). The PCR program consisted of an initial denaturation at 95°C for 3 minutes, 35 cycles of denaturation at 95°C for 20 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 2 minutes, and a final extension at 72°C for 5 minutes. The resultant PCR products were separated by gel electrophoresis (1% agarose), and the 888–base pair fragment (corresponding to SULT1A3 cDNA) was collected and purified. The obtained SULT1A3 cDNA was then subcloned to the pLVX-mCMV-ZsGreen-PGK-Puro vector using the T4 DNA ligase (NEB, Beverly, MA). The recombinant plasmids were transformed into Escherichia coli JM109 cells. After ampicillin selection, several clones were picked up and analyzed for the presence of target cDNA on a 1% agarose gel. The recombinant plasmids in positive colonies were prepared and purified by a Plasmid Maxi preparation kit (Qiagen, Hilden, Germany). The cloned SLULT gene was sequenced within the vector construct [forward primer: CMV-F (5′-CGCAAATGGGCGGTAGGCGTG-3′); reverse primer: pLVX-SULT1A3-BamHI-R (5′-CGCGGATCCTCACAGCTCAGAGCGGAAGCTGAGGCT-3′)].

Lentiviral Vector Production.

Lentiviral vectors were produced by transient transfection 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’s modified 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 were maintained in DMEM containing 10% FBS. On day 4, the cells were cultured in DMEM containing 10% FBS and 6 μg/ml puromycin and the medium was changed every 2 or 3 days. After 1 week, the medium was changed to DMEM containing 10% FBS and 2 μg/ml puromycin. Once 100% confluence was reached, the cells were collected and processed for DNA identification. Stably transfected cells (named SULT293 cells) were obtained after continuous culture for two passages. The transfection efficiency was evaluated by a fluorescence microscopy (Olympus IX71; Olympus Optical Co. Ltd, Tokyo, Japan).

Transient Transfection of shRNA Plasmids

The shRNA plasmids targeting MRP4 have been constructed in our previous study (Quan et al., 2015). The shRNA plasmids were transiently transfected into SULT293 cells as described (Quan et al., 2015). In brief, the SULT293 cells were seeded at a density of 2.0 × 105 cells/well in a six-well plate and maintained at 37°C under 5% CO2 in DMEM containing 10% FBS. On the next day, the plasmid construct carrying the shRNA or scramble (4 μg) was transfected into the cells using Polyfectine according 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 TRIzol extraction method. The total RNA was converted to cDNA using the iScript cDNA synthesis kit according to the manufacturer’s protocol (Bio-Rad, Hercules, CA). The PCR conditions were as follows: 3-minute denaturation at 95°C, followed by 30 cycles of 20 seconds at 95°C, 30 seconds at 55°C, and 30 seconds at 72°C, and a 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 found in our previous publication (Quan et al., 2015). The forward and reverse primers for MRP5 were 5′-GAGAACTCGACCGTTGGAATG-3′ and 5′-TTCGCAGGGAAGCAGCGTCTGG-3′, respectively. The forward and reverse primers for MRP6 were 5′-AAGATGGTGCTTGGATTCGC-3′ and 5′-CAGAGACAGGCATAGGTAGGTGGA-3′, respectively. After reverse transcription–polymerase chain reaction, agarose gel electrophoresis and UV visualization were used to determine the relative amounts of PCR products.

Quantitative Real-Time PCR

Quantitative PCR (qPCR) experiments were performed using the TRIzol extraction method as described in our previous publication (Zhang et al., 2015). In brief, 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 at 72°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 μg cDNA in 10 μl SYBR green/Flourescein qPCR Master Mix (Fermentas, Canada) and 8 pmol of each primer in a final volume of 20 μl. The relative amount of each test mRNA was normalized to the level of glyceraldehyde-3-phosphate dehydrogenase, and the data were analyzed according to the 2–ΔΔCT 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 were euthanized. The livers were collected (4°C) and cut into tiny pieces, followed by suspension in the homogenization buffer. Homogenization was performed with a motorized Teflon/glass homogenizer. After 15-minute centrifugation (9000g) at 4°C, the fat layer and pellet were discarded and the supernatant (i.e., S9 fraction) was collected and stored at −80°C until use.

Preparation of Cell Lysate

SULT293 cells collected in 50 mM potassium phosphate buffer (pH 7.4) were disrupted by sonication for 15 minutes in an ice-cold water bath. Cell lysate was obtained by centrifugation (4°C) at 1000g for 5 minutes. Protein concentration was determined by the Bio-Rad protein assay kit using bovine serum albumin as a standard.

Sulfonation Assay

Sulfonation activities were measured following our published procedures (Meng et al., 2012). In brief, SULT293 cell lysate or expressed SULT1A3 enzyme at a final concentration of 0.1 mg protein/ml was added to 100 μM 3′-phosphoadenosine-5′-phosphosulfate and chrysin/apigenin (at varying concentrations) in a total reaction volume of 200 μl. The mixture was incubated at 37°C for 30 minutes. The reaction was stopped by adding 100 μl of ice-cold acetonitrile. The samples were then centrifuged at 18,000g for 15 minutes and the supernatant was analyzed by ultra performance liquid chromatography (UPLC).

Sulfate Excretion Experiments

The experimental procedures for sulfate excretion were similar to those for glucuronide excretion detailed previously (Quan et al., 2015; Zhang et al., 2015). In brief, the cells were incubated with Hank’s buffered salt solution containing hesperetin (2.5 or 10 μM) at 37°C. Transporter inhibitors, when used, were coincubated with hesperetin. At each time point (0.5, 1, 1.5, and 2 hours), a 200-μl aliquot of incubation medium was sampled and immediately replaced with the same volume of dosing solution. The samples were subjected to UPLC analyses to determine the sulfate concentrations. After sampling at the last time point, the cells were collected and processed to measure the intracellular amounts of sulfate conjugates. The excretion rate of intracellular sulfate was calculated exactly 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 divided by Ci (Ci was the intracellular concentration of sulfate).

A different set of experiments was performed to obtain the hesperetin/sulfates levels (both extracellular and intracellular) versus time profiles for pharmacokinetic modeling as described (Sun et al., 2015). In brief, the cells were incubated with 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 culture wells (n = 3). The cells were processed, and intracellular aglycone and sulfates were measured as described above.

Vesicular Transport Assay

The vesicular transport assay was performed using the rapid filtration technique as described (Wu et al., 2012). In brief, MRP4 membrane vesicles were incubated with H-3′-S or H-7-S in the presence or absence of ATP. Vesicular uptake of sulfate was terminated by the addition of an ice-cold transport buffer, followed by rapid filtration with class F glass fiber filters (pore size: 0.45 μm). Filters were washed, cut, and transferred to a solution of 50% methanol. After sonication for 15 minutes and centrifugation at 18,000g for 15 minutes, the supernatant was collected and subjected to UPLC analysis. ATP-dependent transport was calculated by subtracting the values obtained in the presence of AMP from those in the presence of ATP.

Sulfate Quantification by UPLC Analysis

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

Immunoblotting

Immunoblotting was performed as described (Quan et al., 2015). In brief, the cell lysate (40 μg of total protein) was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (8% acrylamide gels) and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA). Blots were probed with the transporter antibodies (i.e., anti-BCRP, anti-MRP1, anti-MRP2, anti-MRP3, anti-MRP4, anti-MRP5, and anti-MRP6) at a dilution of 1:1000, followed by horseradish peroxidase–conjugated rabbit anti-goat IgG (Santa Cruz Biotechnology, Santa Cruz, CA). Protein bands were visualized by enhanced chemiluminescence.

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. Substrate inhibition refers to the inhibition of enzyme activity at high substrate concentrations. Parameter estimation was performed using Graphpad Prism 5 (San Diego, CA).Embedded Image(1)where Km is the Michaelis constant, Vmax is the maximal velocity, and Ksi is the substrate inhibition constant.

Pharmacokinetic Modeling and Data Fitting

A two-compartment model (Fig. 1), consisting of extracellular and cellular compartments, was established to describe the transport, metabolism, and excretion processes in SULT293 cells. The mass balance equations for the model are shown below (eqs. 2–7). The subscripts m and c denoted the extracellular 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-3′-S (S1) and H-7-S (S2) by SULT1A3 obeyed the substrate inhibition kinetics (Vmax,1, Km,1, and Ksi,1 for S1; Vmax,2, Km,2, and Ksi,2 for S2). Sulfate excretion was a saturable process described by Jmax and K′m (Jmax,1 and K′m,1 for S1; Jmax,2 and K′m,2 for S2). fu denoted the unbound fraction of hesperetin in the cell compartment. The model assumed that binding of sulfates (polar compounds) to cellular proteins was negligible.

Fig. 1.
Fig. 1.

Schematic representation of a two-compartment model that depicts the sulfonation 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-3′-S; S2, H-7-S. The subscripts m and c denote the extracellular and cellular compartments, respectively.

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

Embedded Image(2)Embedded Image(3)Embedded Image(4)Embedded Image(5)Embedded Image(6)Embedded Image(7)

Statistical Analysis

Data are expressed as mean ± S.D. Mean differences between treatment and control groups were analyzed by Student’s t test using Graphpad Prism 5 (San Diego, 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 SULT293 Cell Model.

No metabolites were found in the medium or within the cells after incubation of wild-type HEK293 cells with hesperetin. In contrast, two monosulfates (i.e., H-3′-S and H-7-S) were generated from hesperetin by SULT293 cells (Fig. 2). Further, the excreted amounts of sulfate metabolites increased with the incubation time (Fig. 2). This enabled us to derive the rate of sulfate excretion at different doses of hesperetin. The rates of sulfate excretion significantly increased (P < 0.001) as the dose was increased from 2.5 to 10 μM (Fig. 3A). Increased sulfate excretion was associated with an elevated level of hesperetin within the cells (Fig. 3B). The detection of sulfates (10–39 pmol/mg protein) inside the cells was additional evidence that the SULT293 cells were capable of catalyzing sulfonation reactions (Fig. 3B). The results overall indicated that overexpression of SULT1A3 generated a cell line that was metabolically active only at the sulfonation pathway. Thus, the SULT293 cells were a useful model to investigate the transport and metabolism of drugs and their sulfate metabolites.

Fig. 2.
Fig. 2.

Representative UPLC chromatograms showing that SULT293 cells were active in the generation and excretion of hesperetin sulfates. The UPLC samples were generated after incubation of the cells with hesperetin (10 μM) at different time points.

Fig. 3.
Fig. 3.

Disposition of hesperetin in SULT293 cells at different doses. (A) Excretion rates of hesperetin sulfates at different loading doses. (B) Intracellular amounts of hesperetin 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 bar representing the standard deviation (n = 3).

Sulfonation Kinetics of Hesperetin with SULT293 Cell Lysate and Expressed SULT1A3 Enzyme.

Both SULT293 cell lysate and recombinant SULT1A3 generated two sulfate metabolites (i.e., H-3′-S and H-7-S) from hesperetin. H-3′-S formation mediated by cell lysate followed the substrate inhibition kinetics (Vmax = 0.66 nmol/min per mg; Km = 12.9 μM; and Ksi = 58.1 μM) (Fig. 4A; Table 1). The formation kinetics of H-7-S were also well described by the substrate inhibition model (Vmax = 0.29 nmol/min per mg; Km = 14.8 μM; and Ksi = 49.1 μM) (Fig. 4A; Table 1). Likewise, regiospecific sulfonation of hesperetin by recombinant SULT1A3 obeyed the substrate inhibition kinetics (Vmax = 3.46 nmol/min per mg, Km = 18.5 μM, and Ksi = 37.9 μM for H-3′-S; Vmax = 1.43 nmol/min per mg, Km = 18.4 μM, and Ksi = 36.4 μM for H-7-S) (Fig. 4B; Table 1).

Fig. 4.
Fig. 4.

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

View this table:
TABLE 1

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

Data are represented by mean ± S.E.

The Km and Ksi values of regiospecific sulfonation derived from the cell lysate were similar (P > 0.05) to their corresponding values from the recombinant enzyme (Table 1). This indicated that the sulfonation activity of the cell lysate arose from the SULT1A3 enzyme stably expressed in the cells. However, the Vmax values (closely related to the enzyme concentration) derived from the SULT1A3 enzyme were nearly 5 times those from the SULT293 cell lysate (Table 1). This was not unexpected because the SULT1A3 enzyme was much more concentrated in the recombinant material compared with the cell lysate preparation. Furthermore, both the cell lysate and SULT1A3 enzyme showed a conjugation preference for the 3′-OH group over the 7-OH group (Fig. 4). The intrinsic clearance values (CLint, reflective of catalytic efficiency) of 3′-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 expressed the mRNAs of BCRP, MRP1, MRP4, and MRP5 according to the reverse transcription–polymerase chain reaction (Fig. 5A). Also, the qPCR results showed that the mRNA levels of BCRP and MRP4 were over 10 times those of MRP1 and MRP5 (Fig. 5B). Further, only BCRP and 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 excretion of sulfate metabolites. It was noteworthy that the engineered SULT293 cells showed an identical expression of the transporters (Fig. 5), indicating that transfection of SULT1A3 did not alter transporter expression.

Fig. 5.
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 in HEK293 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.

Effects of MK-571 on Hesperetin Sulfonation.

The effects of MK-571, a pan-MRP inhibitor, on hesperetin sulfonation were determined using the SULT293 cell lysate and the expressed SULT1A3 enzyme. MK-571 at all tested concentrations (5–20 μM) did not show any modulatory effects on hesperetin sulfonation mediated by cell lysate (Fig. 6, A and B). Neither H-3′-S nor H-7-S formation was altered in the presence of MK-571 (Fig. 6, A and B). Likewise, sulfonation of hesperetin by expressed SULT1A3 was not modulated by MK-571 (Fig. 6, C and D). The results indicated that the MRP inhibitor MK-571 had no effects on SULT1A3 activity.

Fig. 6.
Fig. 6.

Effects of MK-571 on sulfonation activity. (A) Effects of MK-571 on sulfonation of hesperetin (2.5 μM) mediated by SULT293 cell lysate. (B) Effects of MK-571 on sulfonation of hesperetin (10 μM) mediated by SULT293 cell lysate. (C) Effects of MK-571 on sulfonation of hesperetin (2.5 μM) mediated by the expressed SULT1A3 enzyme. (D) Effects of MK-571 on sulfonation of hesperetin (10 μM) mediated by the expressed SULT1A3 enzyme. Each data point was the average of three determinations, with the error bar representing the standard deviation (n = 3).

Effects of MK-571 on Excretion of Hesperetin Sulfates.

At a low loading dose (2.5 μM) of hesperetin, MK-571 caused substantial reductions (>57.5%; P < 0.001) in sulfate excretion (Fig. 7A). It also led to marked elevations (>256%; P < 0.001) in sulfate accumulation (i.e., intracellular sulfates) (Fig. 7B). Then, it was not surprising that the apparent efflux clearances (CLef,app) of sulfates were dramatically suppressed (<10.4% of control) in the presence of MK-571 (Fig. 7C). It was noteworthy that MK-571 at 20 μM essentially abolished cellular excretion of both H-3′-S and H-7-S as the sulfate excretion was only 6% of the control (Fig. 7A).

Fig. 7.
Fig. 7.

Effects of MK-571 on sulfate disposition after incubation of SULT293 cells with hesperetin (2.5 μM). (A) Effects of MK-571 on the excretion rates of hesperetin sulfates. (B) Effects of MK-571 on the intracellular levels of hesperetin sulfates. (C) Effects of MK-571 on the efflux clearances (CLef,app) of hesperetin sulfates. Each data point was the average of three determinations, with the error bar representing the standard deviation (n = 3). H-3′-S, hesperetin-3′-O-sulfate; H-7-S, hesperetin-7-O-sulfate. ***P < 0.001 compared with vehicle control.

Similar effects of MK-571 on sulfate disposition were observed when a higher loading dose (10 μM) of hesperetin was used (Fig. 8). MK-571 (5–20 μM) 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 were substantially decreased (>91.8%; P < 0.001) (Fig. 8C). It was clear that the extent of excretion inhibition depended on the inhibitor concentration. A higher extent of excretion inhibition was observed with a higher inhibitor concentration (Figs. 7 and 8). Taken together, our results strongly suggested that the MRP family transporter, more specifically, MRP4 (the only MRP protein expressed in the cells), was an important contributor to the excretion of hesperetin sulfates.

Fig. 8.
Fig. 8.

Effects of MK-571 on sulfate disposition after incubation of SULT293 cells with hesperetin (10 μM). (A) Effects of MK-571 on the excretion rates of hesperetin sulfates. (B) Effects of MK-571 on the intracellular levels of hesperetin sulfates. (C) Effects of MK-571 on the efflux clearances (CLef,app) of hesperetin sulfates. Each data point was the average of three determinations, with the error bar representing the standard deviation (n = 3). ***P < 0.001 compared with vehicle control.

Effects of MRP4 Knockdown on Sulfate Excretion.

MRP4 was knocked down by transient transfection of shRNA. The selected shRNA was shown to significantly decrease the expression of target transporter 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). On the 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 in sulfate excretion caused by decreasing MRP4 expression was additional evidence that MRP4 played a critical role in the excretion of hesperetin sulfates.

Fig. 9.
Fig. 9.

Effects of MRP4 silencing on sulfate disposition. (A) Effects of MRP4 silencing on the excretion rates of hesperetin sulfates. (B) Effects of MRP4 silencing on the intracellular levels of hesperetin sulfates. (C) Effects of MRP4 silencing on the efflux clearances (CLef,app) of hesperetin sulfates. Each data point was the average of three determinations, with the error bar representing the standard deviation (n = 3). Statistically significant differences between scramble and shRNA-treated cells are indicated by asterisks (*P < 0.05; **P < 0.01; and ***P < 0.001).

Transport Kinetics of Hesperetin Sulfates with Human MRP4.

Transport of hesperetin sulfates (H-3′-S and H-7-S) by human MRP4 was investigated using membrane vesicles (Fig. 10). It was clear that hesperetin sulfates were good substrates for transport by human MRP4 (Fig. 10). Transport of both H-3′-S and H-7-S by MRP4 followed Michaelis-Menten kinetics (Jmax = 18.4 pmol/min per mg and K′m = 2.74 μM for H-3′-S; Jmax = 15.5 pmol/min per mg and K′m = 3.24 μM for H-7-S) (Fig. 10). The Jmax and K′m values of H-3′-S were similar (P > 0.05) to the corresponding values of H-7-S, indicating that MRP4 had an equal transport activity toward the positional sulfate isomers (Fig. 10).

Fig. 10.
Fig. 10.

Kinetic profiles for transport of H-3′-S (A) and H-7-S (B) with MRP4 membrane vesicles. The units for K′m and Jmax are μM and pmol/min per mg protein, respectively. Each data point was the average of three determinations, with the error bar representing the standard deviation (n = 3).

Effects of Ko143 on Excretion of Hesperetin Sulfates.

Ko143 is a potent and selective inhibitor of BCRP. However, use of Ko143 (5–20 μM) did not cause any changes in either sulfate excretion or sulfate accumulation (Fig. 11). We also found that Ko143 did not alter the sulfonation of hesperetin by SULT1A3 (data not shown). The results indicated that the role of BCRP in the excretion of hesperetin sulfates was none or negligible, although BCRP was expressed in the cells (Fig. 5).

Fig. 11.
Fig. 11.

Effects of Ko143 on sulfate disposition after incubation of SULT293 cells with hesperetin (10 μM). (A) Effects of Ko143 on the excretion rates of hesperetin sulfates. (B) Effects of Ko143 on the intracellular levels of hesperetin sulfates. Each data point was the average of three determinations, with the error bar representing the standard deviation (n = 3).

Mechanistic Pharmacokinetic Model for Hesperetin/Sulfate Disposition in SULT293 Cells.

The concentration-time profiles were determined for extracellular hesperetin/sulfates and intracellular hesperetin/sulfates at a dose of 5 nmol hesperetin (Fig. 12). A two-compartment pharmacokinetic model (Fig. 1) integrating drug uptake and sulfonation 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 vitro characterization (i.e., Km and Ksi from the sulfonation assay and K′m from the MRP4 vesicular transport assay) (Table 2). The coefficients of variations for fitted parameters were <20%, which is suggestive of adequate fitting (Table 2). Adequate fitting of the model to data helped us to fully understand the cellular deposition processes of hesperetin via the sulfonation pathway (Fig. 12). Following rapid uptake into the cells by passive diffusion, hesperetin was conjugated to form two sulfate isomers (Fig. 13). The generated sulfates were then excreted into the extracellular compartment primary via the action of MRP4 (Fig. 13).

Fig. 12.
Fig. 12.

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

View this table:
TABLE 2

Fitted parameters for sulfonation of hesperetin and efflux of its sulfate metabolites in SULT1A3-overexpressing HEK293 cells

Fig. 13.
Fig. 13.

A summary of disposition processes of hesperetin and its sulfate metabolites in SULT293 cells.

Discussion

In this study, we characterized the sulfonation of hesperetin and excretion of its sulfate metabolites using HEK293 cells overexpressing SULT1A3 (named SULT293 cells). The SULT293 cells were able to sulfate hesperetin at both the 3′-OH and 7-OH groups, owing to stable expression of the SULT1A3 enzyme. The generation and excretion of two sulfates (H-3′-S and H-7-S) enabled us to simultaneously evaluate the excretion of positional isomers of hesperetin sulfates. It was found for the first time that excretion of both sulfate isomers in the cells was primarily contributed by MRP4. The evidence was strong and included four sets of independent results. First, inhibition of sulfate excretion by MK-571 (a selective inhibitor of MRP family proteins) was essentially complete (∼94% inhibition at 20 μM MK-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 for transport by MRP4 according to the vesicular transport assay (Fig. 10). Fourth, the mechanistic pharmacokinetic model, assuming MRP4 as the only sulfate exporter (i.e., the model parameters K′m,1 and K′m,2 values were fixed at those values derived from MRP4 membrane vesicles during 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 importance because 1) these hydrophilic conjugates are the main circulating metabolites; and 2) the conjugates possess many types of biologic activities, such as antioxidant and anti-inflammatory effects (Proteggente et al., 2003; Trzeciakiewicz et al., 2010; Yang et al., 2012; Yamamoto et al., 2013; Gamo et al., 2014). It has been shown that multiple transporters (BCRP, MRP2, and MRP3) are potentially responsible for efflux transport of hesperetin glucuronides (Brand et al., 2008, 2011). Our work was the first report that excretion of hesperetin sulfates was mainly mediated by MRP4 in HEK293 cells. Due to significant expression in the liver and kidney (Ritter et al., 2005; Russel et al., 2008), MRP4 should be an important contributor to hepatic/renal excretion of hesperetin sulfates. Our finding that MRP4 transported sulfate metabolites was consistent with a previous study, in which MRP4 participated in hepatic excretion of the sulfate metabolites of acetaminophen, 4-methylumbelliferone, and harmol (Zamek-Gliszczynski et al., 2006a). However, edaravone sulfate was not transported by MRP4 in the study 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 none or negligible. This was because Ko143 (a potent and selective inhibitor of BCRP) 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 change sulfate disposition (Quan et al., 2015; Zhang et al., 2015). Hence, we were convinced that BCRP was not involved in the efflux transport of hesperetin sulfates. However, this finding appeared to be inconsistent with a previous study of Brand et al. (2008), in which BCRP played a role in the apical efflux of H-7-S in Caco-2 cells. Although data interpretation by Brand et al. (2008) may be confounded by the fact that Ko143 has the potential 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 3′-OH and 7-OH groups, with a positional preference for the former, was consistent with the study of Brand et al. (2010a). In the current work, regiospecific sulfonation of hesperetin by the SULT1A3 and 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 also showed substrate inhibition kinetics. However, Brand et al. (2010a) reported that regiospecific sulfonation of hesperetin by SULT1A3 obeyed Michaelis-Menten kinetics. It remained to be clarified why SULT1A3 behaved differently in the Brand et al. study. Nevertheless, it should be noted that sulfonation of many chemicals (e.g., dopamine, catechin, and eriodictyol) by SULT1A3 displayed the substrate inhibition phenomenon (Wu, 2011).

The wild-type HEK293 cells were unable to conjugate hesperetin due to a lack of expression of SULT1A3. However, HEK293 cells were reported to intrinsically express other types of SULT enzymes, such as SULT1C1 and SULT1E1 (Kapoor et al., 2007; Sheng and Acquaah-Mensah, 2011). Further, the SULT1E1 enzyme shows in vitro conjugation activity toward hesperetin, although the relative activities between SULT1E1 and SULT1A3 were not determined (as the enzyme levels in the recombinant materials were unknown) (Brand et al., 2010a). The exact reason was unknown as to why wild-type HEK293 cells were inactive in conjugating hesperetin. It was hypothesized that the inability of HEK293 cells to metabolize hesperetin was due to a low expression of SULT1E1 in HEK293 cells and/or low sulfonation activity of SULT1E1 toward the compound.

The effects of the transport inhibitor MK-571 on SULT1A3-mediated sulfonation of hesperetin were determined using both the cell lysate preparation and recombinant enzyme (Fig. 6). The information was necessary to accurately interpret the results of sulfate excretion inhibition. This was because modulation of enzyme activity by the transporter inhibitors was a confounding factor to identification of the transporters using the chemical inhibition method (Quan et al., 2015). It was found that MK-571 did not alter the sulfonation rates of hesperetin. Therefore, changes in the excretion profiles of hesperetin sulfates caused by MK-571 were 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 of MRP4 knockdown on sulfate excretion (Fig. 9). The efficiency of protein knockdown highly depended on the shRNA fragments. We have designed four different shRNA sequences for MRP4 and evaluated their performance in knocking down MRP4 in our previous study (Zhang et al., 2015). The best-performing shRNA was obtained based on the interference efficiency and was used to silence MRP4 in the present study. The selected shRNA caused a significant reduction (∼65%) in the target protein MRP4 while showing no effects on the off-target transporters (Zhang et al., 2015).

Sulfonation of hesperetin in vitro favored the formation of H-3′-S over H-7-S (Fig. 4). The formation ratio was about 2.0 at hesperetin concentrations of 2.5 and 10 μM. It was interesting to note that the excretion ratio of H-3′-S over H-7-S in SULT293 cells was close to 2.0 at loading doses of 2.5 and 10 μM hesperetin. An unchanged ratio in the sulfate isomer excretion suggested that the efflux transporter MRP4 had similar transport activities toward H-3′-S and H-7-S. This was supported by vesicular transport assays that showed H-3′-S and H-7-S were equally transported by MRP4, with similar kinetic parameters (P > 0.05). However, it was not always true that the efflux transporters have equal affinities for positional isomers of hesperetin conjugates. For instance, interaction of BCRP with hesperetin glucuronides depended on the position of substitution. Hesperetin 7-O-glucuronide was a high-affinity substrate of BCRP, whereas hesperetin 3′-O-glucuronide was a nonsubstrate 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 of hesperetin and its sulfate metabolites (Fig. 1). Following rapid uptake into the SULT293 cells by passive diffusion, hesperetin was conjugated by SULT1A3 to form two sulfate isomers (Fig. 13). Passive transport of hesperetin across membranes has also been documented in the literature (Brand et al., 2008). The generated sulfates were then excreted into the extracellular compartment by efflux transporter(s), with a dominant contribution from MRP4 (Fig. 13). MRP4 appeared to be a “molecular switch” that controlled cellular efflux of hesperetin sulfates. Thus, inhibition of MRP4 activity or decreasing MRP4 expression led to a reduced sulfate excretion (Figs. 7–9). The results highlighted that the efflux transporter MRP4 played an important role in the disposition of sulfate 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 characterize MRP4-mediated transport of sulfate metabolites (including positional isomers). The SULT293 cells have an advantage over other methods/tools (such as membrane vesicles and monolayer cells overexpressing a transporter) because drug sulfates (usually lacking in commercial availability) were not required for experimentation as the metabolites are generated from the dosed drug by the cells. In addition, the SULT293 cells were free of the concerns raised in transporter identification studies using membrane vesicle or monolayer cells (Fahrmayr et al., 2012). First, drug sulfates poorly cross cellular membranes by passive diffusion. Use of polarized monolayers (expressing a transporter) with administration of the sulfate can be problematic because the sulfate may not enter the cells (Fahrmayr et al., 2012). Second, studies with inside-out vesicles are time consuming and challenging (Fahrmayr et al., 2012).

In summary, two monosulfate metabolites, H-3′-S and H-7-S, were generated from hesperetin in SULT293 cells and efficiently excreted into the extracellular compartment. The pan-MRP inhibitor MK-571 at 20 μM essentially abolished cellular excretion of both H-3′-S and H-7-S, whereas Ko143 had no effects on sulfate excretion. Knockdown of MRP4 led to a substantial reduction (>47.1%) in sulfate excretion. Further, H-3′-S and H-7-S were good substrates for transport by MRP4 according to vesicular transport assay. Taken together, we concluded that MRP4 dominated the excretion of hesperetin sulfates in SULT293 cells. Due to significant expression of MRP4 in various organs/tissues, MRP4 should be a determining factor for the elimination and body distribution of hesperetin sulfates.

Authorship Contributions

Participated 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.

Footnotes

    • Received June 15, 2015.
    • Accepted July 29, 2015.

  • H.S. and X.W. contributed equally to this work

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

  • dx.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
human embryonic kidney
H-3′-S
hesperetin-3′-O-sulfate
H-7-S
hesperetin-7-O-sulfate
Km
Michaelis-Menten constant
Ksi
substrate inhibition constant
MRP
multidrug resistance-associated protein
PCR
polymerase chain reaction
qPCR
quantitative polymerase chain reaction
S1
hesperetin-3′-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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Transport of Hesperetin Sulfates by MRP4

Hua Sun, Xiao Wang, Xiaotong Zhou, Danyi Lu, Zhiguo Ma and Baojian Wu
Drug Metabolism and Disposition October 1, 2015, 43 (10) 1430-1440; DOI: https://doi.org/10.1124/dmd.115.065953
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