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Jun 12, 2015 - In this study, the role of futile recycling (or deglucuronidation) in the disposition of two flavonoids (i.e., genistein and apigenin) ...
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Decreased Expression of Multidrug Resistance-Associated Protein 4 (MRP4/ABCC4) Leads to Reduced Glucuronidation of Flavonoids in UGT1A1-Overexpressing HeLa Cells: The Role of Futile Recycling Hua Sun,∥ Xiaotong Zhou,∥ Xingwang Zhang, and Baojian Wu* Division of Pharmaceutics, College of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, China ABSTRACT: In this study, the role of futile recycling (or deglucuronidation) in the disposition of two flavonoids (i.e., genistein and apigenin) was explored using UGT1A1-overexpressing HeLa cells (or HeLa1A1 cells). Glucuronidation of the flavonoids by HeLa1A1 cell lysate followed the substrate inhibition kinetics (Vmax = 0.10 nmol/min/mg, Km = 0.54 μM, and Ksi = 2.0 μM for genistein; Vmax = 0.19 nmol/min/mg, Km = 0.56 μM, and Ksi = 3.7 μM for apigenin). Glucuronide was efficiently generated and excreted after incubation of the cells with the aglycone (at doses of 1.25−20 nmol). The excretion rates were 0.40−0.69 and 0.84−1.1 nmol/min/mg protein for genistein glucuronide (GG) and apigenin glucuronide (AG), respectively. Furthermore, glucuronide excretion and total glucuronidation were significantly reduced in MRP4 knocked-down as compared to control cells. The alterations were well characterized by a two-compartment pharmacokinetic model incorporating the process of futile recycling (defined by a first-order rate constant, Kde). The derived Kde values were 15 and 25 h−1 for GG and AG, respectively. This was well consistent with the in vitro observation that AG was subjected to more efficient futile recycling compared to GG. In conclusion, futile recycling was involved in cellular glucuronidation, accounting for transporter-dependent glucuronidation of flavonoids. KEYWORDS: glucuronidation, UGT, efflux transporters, MRP4, HeLa cells

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INTRODUCTION Absorption, distribution, metabolism, and excretion (ADME) are the four main processes of in vivo disposition of foreign compounds such as drugs and food-derived chemicals. In fact, poor ADME properties constitute the main causes of high drug attrition rate.1 Hence, determination of the ADME properties of drug candidates has become a major task in drug discovery and development programs.2 The metabolism has received particular attention due to its role in the detoxification, elimination, and pharmacodynamics of xenobiotics/drugs.3,4 Drug-metabolizing enzymes are classified into phase I and II enzymes.5 Phase I enzymes catalyze the oxidation, reduction, and hydrolysis reactions, whereas phase II enzymes catalyze the conjugation reactions such as glucuronidation and sulfonation.6 Although for the majority of xenobiotics phase II metabolism occurs after they are first subjected to phase I reactions, many drugs and dietary polyphenols (e.g., flavonoids) are directly metabolized via phase II metabolism such as glucuronidation.7 Glucuronidation is an important metabolic pathway for numerous xenobiotics and endogenous compounds in humans.8 It is well-accepted that glucuronidation is the principal phase II metabolism because it is responsible for the clearance of ∼35% drugs metabolized by phase II enzymes.9 In glucuronidation reactions, the glucuronic acid moiety (from the cofactor UDPGA) is transferred to the substrates via the action of the UDP-glucuronosyltransferase (UGT) enzymes. In humans, UGT enzymes are divided into five families, namely, UGT1A, UGT2A, UGT2B, UGT3A, and UGT8A.10 The UGT1A and 2B enzymes contribute significantly to metabolism and detoxification of xenobiotics.10 The UGT1A family contains nine members (i.e., UGT1A1, 1A3, 1A4, 1A5, 1A6, 1A7, 1A8, 1A9, and 1A10), whereas the UGT2B family © 2015 American Chemical Society

contains seven (i.e., UGT2B4, 2B7, 2B10, 2B11, 2B15, 2B17, and 2B28). Of note, UGT1A1 is one of the most important members due to its irreplaceable roles in detoxification of bilirubin and SN-38.11,12 Multidrug resistance-associated protein 4 (MRP4/ABCC4) is a member of the C subfamily of ATP-binding cassette (ABC) transporters. By using the energy of ATP binding and hydrolysis, the ABC transporters mediate active transport of their substrates across cell membranes.13 MRP4 is found significantly in various organs/tissues, particularly in the hepatocytes, blood−brain barrier, and proximal tubules of kidney.14 MRP4 is known to transport a variety of xenobiotics including antiviral, antibiotic, cardiovascular, and cytotoxic drugs.14 Hence, like other efflux transporters, MRP4 plays an important role in brain distribution and hepatic/renal excretion of many drugs.15,16 In addition, MRP4 has been implicated in cellular communication and signaling due to its marked ability to transport various signaling molecules (e.g., cyclic nucleotides, urate, and eicosanoids).14 Active transport of glucuronides out of cells is a required step in xenobiotic clearance via UGT metabolism because this type of metabolite with a high hydrophilicity usually lacks the ability of passive transport.17−19 Following generation of glucuronides within the cells, excretion of these metabolites is largely contributed by the efflux transporters (or exporters) including BCRP/ABCG2 (breast cancer resistance protein) and MRP/ ABCC family proteins.17,20 The efflux transporters work in Received: Revised: Accepted: Published: 6001

February 23, 2015 June 5, 2015 June 12, 2015 June 12, 2015 DOI: 10.1021/acs.jafc.5b00983 J. Agric. Food Chem. 2015, 63, 6001−6008

Article

Journal of Agricultural and Food Chemistry

DNA polymerase (TransGen Biotech, Beijing, China). The forward primer was 5′-GTCGCGGGAGTGCAAGGAG-3′, and the reverse primer was 5′-TGTTGATGGCGATAGTGATT-3′. After RT-PCR, agarose gel electrophoresis was performed, and DNA bands were visualized under UV light. Glucuronidation Assay. The aglycones (i.e., genistein and apigenin) were incubated with HeLa1A1 cell lysate to determine the rates of glucuronidation as described in our publications.29−31 In brief, in addition to the aglycone (substrate), the incubation medium contained the lysate (26.5 μg/mL), MgCl2 (0.88 mM), saccharolactone (4.4 mM), alamethicin (22 μg/mL), and UDPGA (3.5 mM) in 50 mM potassium phosphate (pH 7.4). The reaction was terminated by adding ice-cold acetonitrile (containing the internal standard biochanin A). The samples were vortexed and centrifuged at 18000g for 15 min. The supernatant was subjected to UPLC analysis. All experiments were performed in triplicate. Modeling of Enzyme Kinetics. Kinetic parameters were derived by fitting the substrate inhibition equation (eq 1) to the data of reaction rates versus substrate concentrations.32,33 Substrate inhibition refers to inhibition of enzyme activity at high substrate concentrations. Parameter estimation was performed using Graphpad Prism (San Diego, CA, USA).

concert with the UGT enzymes to eliminate foreign compounds from the body.20 Contribution of MRP2 to excretion of glucuronides has been well established.21−24 However, the role of MRP4 in transporting glucuronides remains elusive despite the fact that estradiol 17-β-Dglucuronide is shown to be a substrate for transport by MRP4.25 In our previous study, it was shown that cellular glucuronidation was altered by inhibiting the exporter activity or decreasing exporter expression.26,27 However, the mechanisms underlying the dependence of UGT metabolism on glucuronide efflux remain elusive. The objective of the present study was to explore the role of futile recycling (or deglucuronidation) in the disposition of two flavonoids (i.e., genistein and apigenin) using UGT1A1-overexpressing HeLa cells (or HeLa1A1 cells). Deglucuronidation refers to the βglucuronidase (GUSB)-mediated hydrolysis reaction in which glucuronidated metabolites were converted back to the parent compounds. The pharmacokinetics of both aglycone and glucuronide in HeLa1A1 and MRP4 knocked-down cells were determined and compared after administration of the aglycone. A two-compartment pharmacokinetic model incorporating the deglucuronidation process was established and used to describe the data. Our result revealed that the effect of glucuronide efflux on total glucuronidation was through a futile recycling mechanism.

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Vmax × [S]

V=

(

K m + [S] 1 +

[S] K si

)

(1)

Km is the Michaelis constant, Vmax is the maximal velocity, and Ksi is the substrate inhibition constant. Glucuronide Excretion Experiments. The excretion experiment was performed essentially as described.26,27 In brief, all culture wells were washed twice with 37 °C HBSS (pH 7.4). Then, the cells were incubated at 37 °C with HBSS (2 mL) containing the aglycone (i.e., genistein or apigenin). At each time point (i.e., 0.5, 1, 1.5, and 2 h), a 200 μL aliquot of extracellular medium was sampled and immediately replaced with the same volume of dosing solution. With the addition of 100 μL of acetonitrile (containing the internal standard biochanin A), the concentrations of glucuronides were measured by UPLC. After sampling at the last time point, the medium was rapidly removed by suction. The cells were processed, and intracellular aglycone and glucuronide were measured following our published procedures.27 A different set of experiments was performed to obtain the aglycone/glucuronide levels (both extracellular and intracellular) versus time profiles. The cells in culture wells were incubated with the aglycone at a dose of 2.5 nmol. At each time point (i.e., 10, 20, and 30 min and 1, 1.5, and 2 h), the incubation medium was sampled from the culture wells (n = 3). The residual medium was rapidly removed by suction. The cells were processed, and intracellular aglycone and glucuronide were measured following our published procedures.27 The excretion rate (ER) of intracellular glucuronide was calculated according to eq 2

CHEMICALS AND METHODS

Chemicals. The pLVX-mCMV-ZsGreen-PGK-Puro vector was purchased from BioWit Technologies (Shenzhen, China). The antiGUSB (human β-glucuronidase) and anti-GAPDH antibodies were purchased from Abcam (Cambridge, MA, USA). Uridine diphosphoglucuronic acid (UDPGA), alamethicin, and D-saccharic-1,4-lactone monohydrate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Apigenin and genistein were purchased from Aladdin Reagents (Shanghai, China). Apigenin-7-O-glucuronide (or apigenin glucuronide (AG)) and genistein-7-O-glucuronide (or genistein glucuronide (GG)) were synthesized in our laboratory using rat liver microsomes as described.28 Cell Culture. HeLa1A1 cells and MRP4 knock-down HeLa1A1 cells (referred to as HeLa1A1-MRP4-shRNA cells) were seeded into six-well plates at a density of 4.0 × 105 cells/well and were grown using Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). On day 2 post seeding, the cells were used for the glucuronide excretion experiments. The procedures for establishment of HeLa1A1 and HeLa1A1-MRP4-shRNA cells have been detailed in our previous publications.26,27 In HeLa1A1-MRP4shRNA cells, the protein level of MRP4 was reduced to 35% as compared to HeLa1A1 cells.27 Development of GUSB Transfected HeLa Cells. HeLa cells were stably transfected with the cDNA of human GUSB using the lentiviral approach following our published procedures.26 In brief, the full-length cDNA (1956 bp) of GUSB was PCR-amplified from the commercial pMD18-T plasmid (Sino Biologic Inc., Beijing, China). After purification, GUSB cDNA was ligated into the pLVX-mCMVZsGreen-PGK-Puro vector. The recombinant vector was then incorporated in the generation of lentiviral particles that were used to transduce the HeLa cells. Stably transfected cells were obtained by puromycin selection and named HeLa-GUSB cells. Detection of GUSB mRNA by Reverse TranscriptionPolymerase Chain Reaction (RT-PCR). 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 (BioRad, Hercules, CA, USA). The PCR conditions were as follows: 3 min of denaturation at 95 °C followed by 30 cycles of 20 s at 95 °C, 30 s at 55 °C, and 30 s at 72 °C and a final step of 72 °C for 5 min using Taq

ER = V

dC dt

(2)

where V is the volume of the incubation medium, C is the cumulative concentration of the glucuronide, and t is the incubation time. The fraction of dose metabolized (f met), an indicator of the extent of drug glucuronidation, was calculated exactly as described (eq 3).27

fmet =

excreted glucuronide + intracelluar glucuronide dosed aglycone

(3)

Pharmacokinetic Modeling and Data Fitting. A two-compartment model (Figure 1), consisting of the (incubation/extracellular) medium and the cell compartments, was used to describe the transport and glucuronidation in HeLa1A1 cells. The mass balance equations are shown (eqs 4−7). Transport of the aglycone (A) across the cell membrane occurred with the clearance CLd, representing passive diffusion. Glucuronide (G) formation followed substrate inhibition kinetics described by Vmax, Km, and Ksi. Excretion of glucuronide followed linear kinetics with the first-order rate constant Kef. Similarly, 6002

DOI: 10.1021/acs.jafc.5b00983 J. Agric. Food Chem. 2015, 63, 6001−6008

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The fraction of unbound (fu) values versus genistein concentration was fitted to a Michaelis−Menten-like equation (eq 8) using the Graphpad Prism v5 software fu =

conversion of glucuronide back to the aglycone (futile recycling or deglucuronidation) obeyed the linear kinetics (Kde). f u,a and f u,g denote the unbound fractions of aglycone and conjugate in the cell compartment, respectively. The model assumed (1) glucuronidation was the only metabolic pathway and (2) transport of aglycone across the membrane was a nonsaturable (linear) process. Modeling and data fitting were performed using MATLAB (The Mathworks Inc., Natick, MA, USA). In data fitting, the protein binding of glucuronide was assumed to be unity ( f u,g = 1). Also the Km and Ksi were fixed as the corresponding values derived from the in vitro glucuronidation assays.

dA c CLd CLd = Am − f Ac − dt Vm Vc u,a

(4) fu,a AcVmax VcK m + fu,a Ac +

(fu,a Ac)2

VcK m + fu,a Ac +

RESULTS Binding of Genistein and Apigenin to HeLa1A1 Cell Lysate. Protein binding of genistein to cell lysate was concentration dependent (Figure 2A). The unbound fraction

(6)

fu,a AcVmax (fu,a Ac)2 VcK si

− Kef fu,g Gc − Kdefu,g Gc (7)

Preparation of Cell Lysate. Cells collected in 50 mM potassium phosphate buffer (pH 7.4) were disrupted by sonication for 15 min in an ice-cold water bath. Cell lysate was obtained by centrifugation (4 °C) at 1000g for 5 min. Protein concentration was determined by BioRad protein assay kit using bovine serum albumin as a standard. Protein Binding Assay. Binding of the aglycones (genistein and apigenin) to cell lysate was measured using the rapid equilibrium dialysis system (RED) (Thermo Scientific, Rockford, IL, USA) as described.34 In brief, the sample chamber was filled with 400 μL of the aglycone in HBSS (pH 7.4) containing the cell lysate (0.4 mg/mL). The buffer chamber was filled with 600 μL of blank buffer. The filled RED device was incubated at 100 rpm at 37 °C for 4 h (sufficient for reaching equilibrium) using an Eppendorf E24 incubator shaker (Hamburg, Germany). After incubation, a 50 μL aliquot from each chamber was mixed with 200 μL of acetonitrile, vortexed, and centrifuged for 15 min at 18000g, and the concentration of aglycone in the supernatant was determined by UPLC. The unbound fraction (fu) was calculated using the equation

fu =

(8)

■

(5)

dGm = Kef fu,g Gc dt dGc = dt

+ Kde

VcK si

fu , g Gc

K s + [S]

where f u‑max is the maximal fraction unbound and Ks is the concentration of genistein at half f u‑max. Hydrolysis of Glucuronides by GUSB. The HeLa-GUSB or HeLa cell lysate was incubated with glucuronides (GG and AG) to determine the rates of glucuronide hydrolysis as described.27 The assayed concentration ranges for GG and AG were 2.5−160 and 3.75− 120 μM, respectively. The hydrolysis rate of GUBS was derived by subtracting the hydrolysis rate of HeLa cell lysate from that of HeLaGUSB cell lysate. Quantification of Aglycones and Glucuronides by UPLC Analysis. Concentrations of aglycones and their glucuronides were determined by using a Waters ACQUITY UPLC system equipped with a BEH column (2.1 × 50 mm, 1.7 μm; Waters). 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 0.45 mL/min. The gradient program was 5% B at 0−0.5 min, 5−40% B at 0.5−1.2 min, 40% B at 1.2−1.5 min, 40−95% B at 1.5−2.8 min, 95% B at 2.8−3.3 min, and 95−5% B at 3.3−4 min. The detection wavelengths were 258 and 340 nm for genistein/GG and apigenin/ AG, respectively. Western Blotting. The cell lysate (40 μg of total protein) was analyzed by SDS-PAGE (8% acrylamide gels), and the proteins were transferred onto PVDF membranes (Millipore, Bedford, MA, USA). Blots were probed with the anti-GUSB antibody and the anti-GAPDH antibody (as internal loading control) followed by horseradish peroxidase-conjugated rabbit anti-goat IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Protein bands were detected by enhanced chemiluminescence (ECL). Statistical Analysis. Data are expressed as the mean ± SD (standard deviation). Mean differences between treatment and control groups were analyzed by Student’s t test. The level of significance was set at p < 0.05 (∗) or p < 0.01 (∗∗) or p < 0.001 (∗∗∗).

Figure 1. Schematic representation of a two-compartment model that describes the glucuronidation of flavonoids and excretion of their glucuronides in HeLa1A1 cells. Please refer to the text for the definition of each parameter.

CL CLd dA m = − d Am + f Ac dt Vm Vc u,a

fu‐max [S]

Figure 2. Protein binding of (A) genistein (0.31−10 μM) to HeLa1A1 cell lysate and (B) apigenin (0.31−10 μM) to HeLa1A1 cell lysate. Each data point is the average of three determinations with error bar representing the standard deviation.

(fu) showed a hyperbolic curve with respect to the genistein concentration (Figure 2A). The data were well characterized by a Michaelis−Menten-like equation (eq 8) with a f u‑max value of 0.84 and a Ks value of 0.39 μM. By contrast, protein binding of apigenin to cell lysate was concentration independent, showing an average fu value of 0.37 (Figure 2B). The results suggested that there was a high possibility that genistein and apigenin bound to intracellular proteins once inside the cells.

[S]buffer chamber [S]sample chamber

where [S]buffer chamber and [S]sample chamber are the concentrations of aglycone in the respective chambers at the end of the incubation. 6003

DOI: 10.1021/acs.jafc.5b00983 J. Agric. Food Chem. 2015, 63, 6001−6008

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Journal of Agricultural and Food Chemistry Glucuronidation of Genistein and Apigenin by HeLa1A1 Cell Lysate. The HeLa1A1 cell lysate was able to generate glucuronides from the aglycones genistein and apigenin. Glucuronidation of the two compounds consistently obeyed substrate inhibition kinetics (Vmax = 0.10 nmol/min/ mg, Km = 0.54 μM, and Ksi = 2.0 μM for genistein; Vmax = 0.19 nmol/min/mg, Km = 0.56 μM, and Ksi = 3.7 μM for apigenin) (Figure 3). Compared to genistein, glucuronidation of apigenin

nmol did not alter the intracellular level of AG (Figure 5B). By contrast, the intracellular level of apigenin markedly rose as the loading dose increased (p < 0.001) (Figure 5C). Expression of GUSB in HeLa, HeLa1A1, and HeLa1A1MRP4-shRNA Cells. Expression of GUSB in HeLa and the modified cells was determined at both mRNA and protein levels (Figure 6). The mRNA of GUSB was found significantly in three cell lines (Figure 6A). Also, all three cell lines expressed the GUSB proteins at an identical level (Figure 6B). Clearly, transfection of HeLa cells did not alter protein expression of GUSB. The results suggested that GUBS within the cells may be involved in cellular glucuronidation. Hydrolysis of Glucuronides (GG and AG) by GUSB. The hydrolysis kinetics of GG and AG by the GUSB enzyme were determined and compared. GUSB catalyzed the hydrolysis of two glucuronides at a wide range of concentrations (Figure 7). Determination of the kinetic parameters such Km and Vmax was unlikely because the reaction rates were linearly related to the tested substrate concentrations (Figure 7). However, we were able to estimate the intrinsic clearance (CLint) by performing linear regression. The estimated CLint values were 115 and 151 μL/h/mg for GG and AG, respectively (Figure 7). The CLint value for AG was higher than that for GG (p < 0.05), suggesting that AG was more efficiently hydrolyzed by GUSB compared to GG. Disposition of Genistein in HeLa1A1 versus HeLa1A1MRP4-shRNA Cells. The concentration−time profiles were determined for extracellular genistein and GG and for intracellular genistein and GG in control versus MRP4 knocked-down (shRNA-MRP4) cells at a dose of 2.5 nmol of genistein (Figure 8). Knock-down of MRP4 led to a substantial decrease in excretion of GG (30.2%, p < 0.01) (Figure 8A) and in the f met value (29.6%, p < 0.01) (Figure 8A,B). A reduction in cellular glucuronidation caused by MRP4 silencing was accompanied by elevations in both extracellular and intracellular levels of the aglycone (genistein) (Figure 8C,D). The two-compartment pharmacokinetic model featuring a futile recycling or deglucuronidation process (Kde) was well fitted to the data (Table 1 and Figure 8). The derived Kef values were 20 and 9.1 1/h for control and shRNA-MRP4 cells, respectively (Table 1). A significant decrease (54.5%, p < 0.01) in the rate constant for glucuronide efflux was well consistent with decreased expression of MRP4 due to gene silencing.

Figure 3. Kinetic profile of (A) glucuronidation derived from incubation of genistein with HeLa1A1 cell lysate and (B) glucuronidation derived from incubation of apigenin with HeLa1A1 cell lysate. Each data point is the average of three determinations with error bar representing the standard deviation.

showed a higher intrinsic clearance value (CLint = Vmax/Km). This suggested that apigenin was glucuronidated more efficiently by cell lysate. Excretion of GG and AG in HeLa1A1 Cells. Genistein glucuronide (GG) was efficiently generated and excreted after incubation of the aglycone (loading doses of 1.25−20 nmol) with the cells. The excretion rates of GG ranged from 0.40 to 0.69 nmol/min/mg. Of note, the excretion rate of GG decreased (p < 0.05) significantly with an increase in the loading dose of genistein (Figure 4A). In response to dose escalation, the intracellular level of GG also decreased (p < 0.05), whereas the intracellular level of genistein increased (p < 0.01) (Figure 4B,C). Likewise, apigenin glucuronide (AG) was efficiently generated and excreted after incubation of the aglycone (loading doses of 1.25−20 nmol) with the cells. The excretion rates ranged from 0.84 to 1.1 nmol/min/mg. Interestingly, the excretion rate of AG was highest at the intermediate dose of 5 nmol (Figure 5A). Dose escalation from 1.25 to 5 nmol caused a substantial increase in the intracellular level of AG (p < 0.001) (Figure 5B). However, further increase of the dose from 5 to 20

Figure 4. Disposition of genistein in HeLa1A1 cells at different doses: (A) excretion rates of genistein glucuronide (GG) at different loading doses; (B) intracellular amounts of GG at 2 h under different loading doses; (C) intracellular amounts of genistein at 2 h under different loading doses. Each data point is the average of three determinations with error bar representing the standard deviation (n = 3). (∗) p < 0.05; (∗∗) p < 0.01; (∗∗∗) p < 0.001. 6004

DOI: 10.1021/acs.jafc.5b00983 J. Agric. Food Chem. 2015, 63, 6001−6008

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Figure 5. Disposition of apigenin in HeLa1A1 cells at different doses: (A) excretion rates of apigenin glucuronide (AG) at different loading doses; (B) intracellular amounts of AG at 2 h under different loading doses; (C) intracellular amounts of apigenin at 2 h under different loading doses. Each data point is the average of three determinations with error bar representing the standard deviation (n = 3). (∗) p < 0.05; (∗∗∗) p < 0.001.

Figure 6. Expression of GUSB in wild-type HeLa, HeLa1A1, and HeLa1A1-MRP4-shRNA cells: (A) mRNA expression of GUSB in wild-type HeLa and modified cells (lanes: 1, detection of GUSB mRNA in wild-type HeLa cells; 2, detection of GUSB mRNA in HeLa1A1 cells; 3, detection of GUSB mRNA in HeLa1A1-MRP4shRNA cells); (B) Western blots of the cell lines against GUSB.

Figure 8. Pharmacokinetic profiles for genistein disposition in HeLa1A1 and shRNA-MRP4 transfected cells at the dose of 2.5 nmol: (A) extracellular GG−time profile; (B) intracellular GG−time profile; (C) extracellular genistein−time profile; (D) intracellular genistein−time profile. Solid lines are predicted data from the pharmacokinetic model depicted in Figure 1.

Disposition of Apigenin in HeLa1A1 versus HeLa1A1MRP4-shRNA Cells. The concentration−time profiles were determined for extracellular apigenin and AG and for intracellular apigenin and AG in control versus shRNA-MRP4 cells at a dose of 2.5 nmol of apigenin (Figure 9). As observed for genistein, MRP4 silencing resulted in significant decreases in excretion of AG (30.5%, p < 0.01) (Figure 9A) and in the f met value (27.7%, p < 0.01) (Figure 9A,B). Reduced glucuronidation in the shRNA-MRP4 cells was accompanied by elevations in both extracellular and intracellular levels of the aglycone (apigenin) (Figure 9C,D). Similarly, the data were well described by the two-compartment pharmacokinetic model incorporating the futile recycling process (Table 1 and Figure 9). The derived Kef values were 23 and 12 1/h for control and shRNA-MRP4 cells, respectively (Table 1). A significant reduction (47.8%, p < 0.01) in the efflux rate constant was well consistent with a compromised efflux capacity due to decreased expression of MRP4.

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Figure 7. Hydrolysis of genistein glucuronide (A) and apigenin glucuronide (B) by GUSB enzyme. Each data point is the average of three determinations with error bar representing the standard deviation.

DISCUSSION In this study, the pharmacokinetics of both flavonoid (genistein or apigenin) and generated glucuronide in HeLa1A1 and MRP4 knocked-down cells were determined and compared after administration of the flavonoid. Consistent with our 6005

DOI: 10.1021/acs.jafc.5b00983 J. Agric. Food Chem. 2015, 63, 6001−6008

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Journal of Agricultural and Food Chemistry

Table 1. Fitted Parameters for Glucuronidation of Two Flavonoids and Efflux of Their Glucuronides in HeLa1A1 Cells genistein parameter Km (μM) Ksi (μM) Vmax (pmol/min) f u,a CLd (mL/h) Kde (1/h) Kef (1/h) a

control cells a

0.54 2.0a 33 ± 2.5 0.040 ± 0.0048 13 ± 3.3 15 ± 2.8 20 ± 2.9

apigenin

MRP4 knockdown cells same as control same as control same as control same as control same as control same as control 9.1 ± 0.23

control cells a

0.56 3.7a 58 ± 3.1 0.023 ± 0.0030 94 ± 7.4 25 ± 3.6 23 ± 1.3

MRP4 knockdown cells same as control same as control same as control same as control same as control same as control 12 ± 1.3

Assigned values from in vitro metabolism kinetics.

with an increase in the substrate concentration (Figure 3A). Likewise, a decrease in the rate of AG excretion when loading apigenin was increased from 5 to 20 nmol (i.e., from 2.5 to 10 μM in terms of incubation concentration) can be well explained by the substrate inhibition kinetics wherein the glucuronidation rate at 10 μM was smaller than that at 2.5 μM (Figures 5A and 3B). In vitro protein binding assays showed that genistein and apigenin bound significantly to cell lysate at a high protein concentration of 0.4 mg/mL, indicating that intracellular binding of the two compounds to proteins may be substantial (Figure 2). This was well consistent with the low values of derived fu values (0.040 and 0.023 for genistein and apigenin, respectively) from pharmacokinetic modeling (Table 1). On the other hand, the derived kinetic parameters for glucuronidation reactions were not corrected using protein binding (Figure 3). This was because binding of the aglycones (log P = 1.7−2.7) to cell lysate (at a low protein concentration of 26.5 μg/mL) in the glucuronidation assays was negligible (fu > 0.99) based on the Hallifax and Houston model. This model has been shown to accurately predict the fu values for the compounds with intermediate lipophilicity.37 Flavonoids are a large class of compounds with many types of health benefits such as anticancer and anti-inflammatory effects.38−40 However, extensive glucuronidation is a significant barrier to the bioavailability of flavonoids, limiting their therapeutic uses. Therefore, a better understanding of the factors that govern glucuronidation of flavonoids would contribute to solving the problem of low bioavailability. The current study and our previous ones showed that reduced glucuronidation of flavonoids was attained by inhibiting exporter activity and decreasing exporter expression.26,27 Hence, it represented a promising approach to modulate the bioavailability of flavonoids by manipulating the activities of efflux transporters such as MRP4. Disposition of genistein and apigenin in HeLa1A1 cells (and MRP4 knocked-down cells) was adequately characterized by the two-compartment pharmacokinetic model (Figure 1). The pharmacokinetic model assumed that the aglycones passed the cell membranes freely by passive diffusion. This assumption was valid as passive diffusion was the predominant mechanism for transport of the two flavonoids.41,42 The model also assumed that efflux of glucuronides followed a linear (nonsaturable) kinetics described by the first-order rate constant Kef. The linear transport kinetics appeared to be valid because saturation of the efflux transporters was not observed for intracellular glucuronides of up to 60 pmol/mg protein (data not shown). In addition, protein binding of glucuronides was not considered (i.e., f u,g = 1) in data fitting; this was reasonable as binding of

Figure 9. Pharmacokinetic profiles for apigenin disposition in HeLa1A1 and shRNA-MRP4 transfected cells at the dose of 2.5 nmol: (A) extracellular AG−time profile; (B) intracellular AG−time profile; (C) extracellular apigenin−time profile; (D) intracellular apigenin−time profile. Solid lines are predicted data from the pharmacokinetic model depicted in Figure 1.

previous study,27 knock-down of MRP4 led to significant reductions in glucuronide excretion and total glucuronidation (representing one aspect of glucuronidation−efflux interplay). In addition, the pharmacokinetic data (in the control versus shRNA-MRP4 cells) were well described by a two-compartment model incorporating the futile recycling process (characterized by the rate constant Kde) (Figures 8 and 9). Furthermore, we showed that the GUSB enzyme was expressed in HeLa and the modified cells (Figure 6). Also, GUSB enzyme was capable of catalyzing efficient hydrolysis of the flavonoid glucuronides back to their parent compounds. Taken together, our results indicated that the futile recycling (enabled by GUSB-mediated deglucuronidation) was essential for the occurrence of glucuronidation−efflux interplay. Glucuronidation of the two model flavonoids (i.e., genistein and apigenin) followed substrate inhibition kinetics (Figure 3). It should be noted that glucuronidation of many other flavonoids by UGT1A1 displays substrate inhibition.32,35,36 The mechanisms for substrate inhibition in glucuronidation reactions remain elusive.32 Nevertheless, the substrate inhibition may be attributable to an allosteric (inhibitory) binding site within the enzyme and/or formation of a ternary dead-end enzyme complex.32 The substrate inhibition kinetics can be used to explain why the excretion rates of GG decreased as the loading dose of genistein increased (Figure 4A). The loading dose ranged from 1.25 to 20 nmol, corresponding to an incubation concentration range of 0.625−10 μM. Within the range of 0.625−10 μM, the rate of glucuronidation decreased 6006

DOI: 10.1021/acs.jafc.5b00983 J. Agric. Food Chem. 2015, 63, 6001−6008

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Journal of Agricultural and Food Chemistry Funding

polar glucuronides to cellular proteins was negligible or nonexistent. Knock-down of the efflux transporter MRP4 leading to reduced glucuronidation of flavonoids represented one aspect of glucuronidation−efflux interplay, as also noted previously.27 In the current study, we provided the first experimental evidence that the futile recycling (deglucuronidation) enabled by GUSB was necessary for this interplay of glucuronidation with glucuronide efflux to occur. Futile recycling appeared to bridge the two processes (i.e., glucuronide formation and excretion), making the interplay of glucuronidation−efflux feasible.19 Impaired glucuronide efflux (due to decreased transporter expression) led to an elevation in the intracellular level of glucuronide (Figures 8 and 9). A rise in intracellular glucuronide was associated with an increase in the rate of futile recycling according to the first-order kinetics (the conversion rate = Kde × intracellular glucuronide) (Figure 1). As a consequence, total formation of glucuronide was altered (i.e., reduced) in the cells. On the contrary, in the absence of futile recycling, glucuronide formation was independent of its downstream process excretion; thus, impact of glucuronide excretion on its formation was impossible.19 Use of HeLa1A1 cells (and transporter knock-down cell lines) for glucuronide transport studies was more advantageous compared to the membrane vesicles or the monolayer cells (e.g., MDCK) overexpressing a single transporter. This was because the HeLa1A1 cells expressing an array of transporters were a closer mimic of the in vivo situations. In addition, synthesis of glucuronide (lacking in commercial availability) was avoided as glucuronide can be generated from dosed aglycone by the cells. Moreover, the HeLa1A1 cells were free of concerns raised in identification studies of glucuronide transporters using membrane vesicle or monolayer cells.22 First, drug glucuronides poorly cross cellular membranes by passive diffusion. Therefore, use of polarized monolayers (expressing a transporter) with administration of glucuronide can be problematic. This was because the glucuronide will not enter the cells.22 Second, studies with inside-out vesicles are time-consuming and challenging.22 In summary, glucuronidation of two flavonoids (i.e., genistein and apigenin) and excretion of their glucuronides were investigated using HeLa1A1 cells. Glucuronidation of these two flavonoids by cell lysate consistently followed substrate inhibition kinetics. GG or AG was efficiently generated and excreted after incubation of the aglycone with the cells. Knockdown of MRP4 resulted in a substantial reduction in the excretion of GG and AG. A significant reduction was also observed for cellular glucuronidation of genistein and apigenin. The alterations in glucuronide excretion and cellular glucuronidation were well characterized by a two-compartment pharmacokinetic model incorporating the process of futile recycling. This study provided the first experimental evidence that the effect of glucuronide efflux on total glucuronidation was through a futile recycling mechanism.

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This work was supported by the Natural Science Foundation of Guangdong Province (Grant 2014A030306014). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED BCRP/Bcrp breast cancer resistance protein DMEM Dulbecco’s modified Eagle’s medium FBS fetal bovine serum f u‑max maximal fraction unbound GUSB β-glucuronidase Km Michaelis−Menten constant; Ks concentration of genistein at half f u‑max Ksi substrate inhibition constant; MRP multidrug resistance-associated protein MS mass spectroscopy P-gp P-glycoprotein QTOF quadrupole time-of-flight UDPGA uridine diphosphoglucuronic acid UGT UDP-glucuronosyltransferase UPLC ultra performance liquid chromatography Vmax maximal velocity

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AUTHOR INFORMATION

Corresponding Author

*(B.W.) Phone: (+86) 020-8522-0482. Fax: (+86) 020-85220482. E-mail: [email protected]. Author Contributions ∥

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