Regioselective glucuronidation of diosmetin and chrysoeriol by the interplay of glucuronidation and transport in UGT1A9-Overexpressing hela cells

Article abstract of DOI:10.1371/journal.pone.0166239

This study aimed to determine the reaction kinetics of the regioselective glucuronidation of diosmetin and chrysoeriol, two important methylated metabolites of luteolin, by human liver microsomes (HLMs) and uridine-50 -diphosphate glucuronosyltransferase

Full text of DOI:10.1371/journal.pone.0166239

RESEARCH ARTICLE
Regioselective Glucuronidation of Diosmetin
and Chrysoeriol by the Interplay of
Glucuronidation and Transport in UGT1A9-
Overexpressing HeLa Cells
Xuejun Zeng1,2☯, Jian Shi2☯, Min Zhao2, Qingwei Chen1,2, Liping Wang2, Huangyu Jiang2,
Feifei Luo2, Lijun Zhu2, Linlin Lu2, Xinchun Wang1*, Zhongqiu Liu2*
1 Department of Pharmacy, First Hospital Affiliated to Shihezi University, Shihezi, Xinjiang, 832002, China,
2 International Institute for Translational Chinese Medicine, Guangzhou University of Chinese Medicine,
Guangzhou, Guangdong, 510006, China
a11111
☯ These authors contributed equally to this work.
* liuzq@gzucm.edu.cn (ZL); cwjwxc@163.com (XW)
Abstract
OPEN ACCESS
This study aimed to determine the reaction kinetics of the regioselective glucuronidation of
diosmetin and chrysoeriol, two important methylated metabolites of luteolin, by human liver
microsomes (HLMs) and uridine-50-diphosphate glucuronosyltransferase (UGTs) enzymes.
This study also investigated the effects of breast cancer resistance protein (BCRP) on the
efflux of diosmetin and chrysoeriol glucuronides in HeLa cells overexpressing UGT1A9
(HeLa—UGT1A9). After incubation with HLMs in the presence of UDP-glucuronic acid,
diosmetin and chrysoeriol gained two glucuronides each, and the OH—in each B ring of
diosmetin and chrysoeriol was the preferable site for glucuronidation. Screening assays
with 12 human expressed UGT enzymes and chemical-inhibition assays demonstrated that
glucuronide formation was almost exclusively catalyzed by UGT1A1, UGT1A6, and
UGT1A9. Importantly, in HeLa—UGT1A9, Ko143 significantly inhibited the efflux of diosme-
tin and chrysoeriol glucuronides and increased their intracellular levels in a dose-dependent
manner. This observation suggested that BCRP-mediated excretion was the predominant
pathway for diosmetin and chrysoeriol disposition. In conclusion, UGT1A1, UGT1A6, and
UGT1A9 were the chief contributors to the regioselective glucuronidation of diosmetin and
chrysoeriol in the liver. Moreover, cellular glucuronidation was significantly altered by inhibit-
ing BCRP, revealing a notable interplay between glucuronidation and efflux transport. Dios-
metin and chrysoeriol possibly have different effects on anti-cancer due to the difference of
UGT isoforms in different cancer cells.
Citation: Zeng X, Shi J, Zhao M, Chen Q, Wang L,
Jiang H, et al. (2016) Regioselective
Glucuronidation of Diosmetin and Chrysoeriol by
the Interplay of Glucuronidation and Transport in
UGT1A9-Overexpressing HeLa Cells. PLoS ONE
11(11): e0166239. doi:10.1371/journal.
pone.0166239
Editor: Alberto G Passi, University of Insubria,
ITALY
Received: June 10, 2016
Accepted: October 25, 2016
Published: November 10, 2016
Copyright: © 2016 Zeng et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper.
Funding: This research was supported by the joint
fund set up by the National Natural Science
Foundation of China (No. U1203204), the National
Natural Science Foundation of China (No.
81460638, 81360671), Science and Technology
Project of Guangzhou City (201509010004) and
Guangdong Natural Science Foundation Province
(2015AD030312012).
Introduction
Luteolin (30,40,5,7-tetrahydroxyflavone), a typical catecholic flavonoid, is present in various
dietary sources such as fruits, vegetables, wines, oils, tea etc [1]. It exhibits a wide range of
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Glucuronidation and Transportation of Diosmetin and Chrysoeriol
Competing Interests: The authors have declared
pharmacological effects, including antioxidation, anti-inflammation, and anticarcinogenic
activity [2,3]. A study reported that after luteolin’s oral administration, it undergoes methyla-
tion as a major metabolic pathway. Isomers diosmetin (5,7,30-trihydroxy-40-methoxyflavone)
and chrysoeriol (5,7,40-trihydroxy-30- methoxyflavone) are the two methylated metabolites [4]
of luteolin. These isomers have been shown to exhibit common biological effects, such as oste-
oporosis prevention [5]. Diosmetin is an active ingredient of some medications [6] and has
been reported to display several biological properties, including anticancer effects [7,8] and
antibacterial actions [9]-. Similar to diosmetin, chrysoeriol is mainly distributed in many plant
products, such as parsley [10] and peanut hull [11]. Chrysoeriol could inhibit lipid peroxida-
tion in low-density lipoprotein [11] and exhibit antioxidant activity and free radical scavenging
ability [12]. The biological properties of flavone are well known to be severely limited by the
compound’s low bioavailability resulting from extensive metabolism and excretion. However,
studies on the mechanism and characteristics of the metabolism and excretion of diosmetin
and chrysoeriol are few.
that no competing interests exist.
Uridine-50-diphosphate glucuronosyltransferases (UGTs) are a superfamily of enzymes that
catalyze the glucuronidation of many compounds [13]. Glucuronidation, a primary phase II
conjugation reaction, is considered as a detoxification mechanism because the generated glu-
curonides are highly polar and can be rapidly eliminated [14,15]. UGT isoforms involved in
glucuronidation of phenolics and other relevant compounds discussed belong to UGT1A or
UGT2B family [13], and these enzymes possess broad and overlapping substrate specificities.
Our previous study demonstrated that diosmetin and chrysoeriol could be metabolized to
their phase II metabolites by Ugts [16]. Thus, the UGT-catalyzed glucuronidation could play a
key role in determining the bioavailability and clearance of diosmetin and chrysoeriol [13].
However, the characteristics of and difference between the glucuronidation of diosmetin and
chrysoeriol by UGTs and the other major contributing enzymes have not been fully estab-
lished. This knowledge is valuable in achieving better prediction of diosmetin and chrysoeriol
disposition, which could be the main factors affecting the compounds’ bioavailability and bio-
logical activities. Our findings could also add to the general understanding of the mechanisms
of action of diosmetin and chrysoeriol in vivo.
On the other hand, the high polarity of the glucuronides renders difficult their passive diffu-
sion outward from the cell. As such, the glucuronides require efflux transporters to exit the
cells. The cellular glucuronidation of flavone is hence presumed to be affected by the activities
of glucuronidation enzymes, relevant efflux transporters, and the interplay between UGT
enzymes and efflux transporters [17]. Breast cancer resistance protein (BCRP), an important
efflux pump in the cell membrane, is known to participate in the biliary and intestinal excretion
of many flavone glucuronides [18–20]. Therefore, the interplay between BCRP and UGT
enzymes could play a key role in the phase II disposition of flavone. Understanding the role of
BCRP is important for assessing the contribution of BCRP in glucuronide disposition. Our
group studied the effect of such interplay on luteolin disposition using established HeLa—
UGT1A9 cells with UGT1A9 and BCRP to investigate the metabolism of luteolin and the excre-
tion of its glucuronides [13]. Compared with Caco-2 and MDCK II cells, HeLa—UGT1A9 cells
serve as a suitable model for the study of the interplay between transporters and UGT enzymes
because of the functional expression of only one dominant UGT and one dominant efflux
transporter in these cells [21,22]. Diosmetin and chrysoeriol are the main methylation metabo-
lites of luteolin. Hence, the study of the effect of the interplay between transporters and UGT
enzymes on diosmetin and chrysoeriol metabolism is important and useful for determining the
mechanism and characteristics of the absorption and metabolism of luteolin in vivo.
The present study aimed to determine and compare the reaction kinetics of the regioselec-
tive glucuronidation of diosmetin and chrysoeriol by human liver microsomes (HLMs) and
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Glucuronidation and Transportation of Diosmetin and Chrysoeriol
expressed UGT enzymes. The main UGT enzymes involved in such regioselective glucuroni-
dation were identified and determined through incubation with a panel of 12 UGT enzymes
and HLMs in the presence of specific UGT inhibitors. Kinetic parameters were derived by fit-
ting and appropriate modeling of the data. UGT1A9 was one of important UGT enzymes in
the liver involved in the metabolism of diosmetin and chrysoeriol as determined in this study.
Hence, we used HeLa—UGT1A9 cells to explore how the interplay between UGT1A9 and
BCRP affects diosmetin and chrysoeriol disposition. The results of our study would contribute
to improving the understanding on the absorption and disposition of luteolin.
Material and Methods
Chemicals and Reagents
Diosmetin (>98% purity) was purchased from Chengdu Must Pharmaceutical Co. Ltd.
(Chengdu, China). Chrysoeriol was acquired from Shenzhen Zhenqiang Bio-Technology Co.
Ltd. (Shenzhen, China). Tilianin (95% purity; internal standard; IS) was provided by the
Department of Pharmacy, First Hospital Affiliated to Shihezi University. Diosmetin-7-O-glu-
curonide (Dio-7-G), diosmetin-30-O-glucuronide (Dio-30-G), chrysoeriol-7-O-glucuronide
(Chr-7-G) and chrysoeriol-40-O-glucuronide (Chr-40-G) were prepared and identified by our
laboratory [16]. Carvacrol and troglitazone were purchased from Qiyun Bio-Technology Co.
Ltd. (Guangzhou, China). Phenylbutazone, bilirubin, UDP glucuronic acid (UDPGA), ala-
methicin, MgCl2, D-saccharic-1,4-lactone monohydrate, β-glucuronidase (Type HP-2 from
Helix pomatia), Hanks’ Balanced Salts, HEPES (N-(2-Hydroxyethyl) piperazine-N’-(2-ethane-
sulfonic acid)), MTT and Ko143 were purchased from Sigma—Aldrich Co. (St. Louis, MO,
USA). Human expressed UGTs (Supersomes Enzymes), pooled HLMs (35 individuals), and
human expressed UGT enzymes (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7,
2B15, and 2B17) were purchased from BD Biosciences (Woburn, MA, USA). HeLa cells and
HeLa cells stably transfected with UGT1A9 (HeLa—UGT1A9 cells) were provided by Dr.
Ming Hu (Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy,
University of Houston, Houston, Texas, USA). HyClone fetal bovine serum and HyClone pen-
icillin—streptomycin solution were obtained from Thermo Fisher Scientific (MA, USA). All
other chemicals and solvents were of analytical grade or better.
Cell Culture
HeLa and HeLa—UGT1A9 cells were seeded into six-well plate at a density of 1.0 × 105
cells/well, and were grown using Dulbecco’s modified Eagle’smedium (DMEM) supplemented
with 10% fetal bovine serum (FBS). After 3 ~ 4 days of seeding, the cells can be used for the
glucuronidation experiments.
Glucuronidation Assay
All metabolism studies in vitro were approved by the First Hospital Affiliated to Shihezi Uni-
versity Research Ethics Committee. The glucuronidation assay was performed as described in
previous publications [23–25]. The stock solutions of diosmetin or chrysoeriol were prepared
in methanol-dimethyl sulfoxide (DMSO) (1:1, v/v) and diluted with methanol—DMSO (1:1,
v/v) to the desired concentrations immediately before use. The incubation volumes were
120 μL, and the total organic solvent content was 1%. The mixtures contained HLMs or
expressed UGT enzymes, MgCl2 (0.88 mM), saccharolactone (4.4 mM), alamethicin
(22 μg/mL), and diosmetin or chrysoeriol (at desired concentrations) in 50 mM potassium
phosphate (pH 7.4). The reactions were initiated by adding UDPGA (3.5 mM) at 37˚C and
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Glucuronidation and Transportation of Diosmetin and Chrysoeriol
then allowed to stand for 30 min in a water bath. These reactions were terminated by adding
60 μL ice-cold acetonitrile containing the IS (25 nM tilianin). The samples were centrifuged at
13,000 rpm for 30 min, and the supernatant was analyzed by ultra high pressure liquid chroma-
tography (UHPLC)–tandem mass spectrometry (MS/MS). All experiments were performed in
triplicate.
Kinetic Evaluation
The enzyme kinetic parameters were obtained by fitting kinetic models to the experimental
data using GraphPad Prism version 5.04 for Windows (GraphPad Software Inc., San Diego,
CA, USA). The best model was selected based on visual inspection of the Eadie-Hofstee plots,
the calculated r2 values, and corrected Akaike’s information criterion [26]. The HLMs or
human expressed UGT enzymes were employed for kinetic studies at concentrations ranging
from 0.0021 mg/mL to 0.0053 mg/mL. The rates of the glucuronidation were fitted to the fol-
lowing equations:
Cmetabolite
t  Cprotein
V ¼
Where V (nmol/mg/min) is the rates of the glucuronidation, Cmetabolite (μM) and Cprotein
(mg/mL) respectively are the concentration of the metabolite and the reaction protein, t (min)
is the reaction time [2,22].
Model selection was based on visual inspection of Eadie—Hofstee plots [26] and it was
reported in our previous study [25]. In brief, if the Eadie—Hofstee plot was linear, formation
rates (V) of glucuronides at different substrate concentrations (C) were fitted to the standard
Eq (1):
Vmax  C
V ¼
ð1Þ
Km þ C
where Km is the Michaelis-Menten constant and Vmax is the maximum rate of glucuronidation.
The intrinsic clearance (CLint) was derived by Vmax/Km.
When Eadie—Hofstee plots revealed substrate inhibition kinetics, the reaction rate (V)
were fitted to Eq (2):
Vmax1
V ¼
ð2Þ
1 þ ðKm1=CÞ þ ðC=KsiÞ
where C is the substrate concentration, V is the initial reaction rate, Vmax1 is the maximum
enzyme velocity, Km1 is the substrate concentration required to achieve 50% of Vmax1, and Ksi
is the substrate inhibition constant. The intrinsic clearance (CLint) was derived by Vmax1/Km1
.
Assay with Human Expressed UGTs
The method of this study as described in detail previously [25]. In this study, 12 human
expressed UGTs (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17)
were used for the glucuronidation of diosmetin and chrysoeriol. Three substrate concentra-
tions (1.25, 2.5, and 10 μM) were employed in this experiment, and the incubation progress
was performed as shown above. The incubation conditions were as follows: protein concentra-
tion: 0.0053 mg/mL; temperature of water: 37˚C; shaker speed: 50 rpm; reaction time: 30 min.
After incubation, all samples were pretreated as described above and analyzed by UHPLC—
MS/MS.
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Glucuronidation and Transportation of Diosmetin and Chrysoeriol
Chemical Inhibition Studies
Bilirubin (UGT1A1 inhibitor)[12,27], phenylbutazone (UGT1A6 inhibitor)[25,28], or carva-
crol (UGT1A9 inhibitor)[17,29] were used to determine glucuronidation activities of diosme-
tin and chrysoeriol in the HLMs and expressed UGTs (UGT1A1, UGT1A6, and UGT1A9).
The protein concentrations of the HLMs and expressed UGTs were 0.0021–0.0106 mg pro-
tein/mL. All of the incubations were run at 37˚C for 30 min, and the way of pretreated and
determined samples were as discribed above.
Glucuronides Excretion Experiments in the HeLa and HeLa—UGT1A9
Cell Model
Glucuronides excretion experiments were performed as described in detail previously [17].
The HeLa cells were used to incubate diosmetin and chrysoeriol as the control. Brifely speak-
ing, the cells were incubated at 37˚C with loading solution (2 mL). The loading solution were
HBSS (9.801 g Hanks’ balanced salts, 0.372 g NaHCO3, 3.502 g glucose, 5.963 g HEPES and
1.164 g NaCl dissolved in 900 mL deionized water) which containing different diosmetin or
chrysoeriol concentrations (1.25, 2.5, 5, 10, 20, and 40 μM with 0.1% DMSO) at the pre-set
time, 500 μL of incubation medium from each well was collected, and the same volume of dios-
metin or chrysoeriol solution was loaded in each well. Ko143, whenever applied, a chemical
inhibitor of BCRP (Ki < 1 μM [30]), was used to determine whether the diosmetin or chry-
soeriol glucuronides were the substrates of BCRP. Ko143 (5 or 10 μM) was diluted in a solution
containing diosmetin or chrysoeriol (2.5 μM). The collected samples were mixed with 250 μL
acetonitrile containing 25 nM tilianin and then analyzed by UHPLC—MS/MS after centrifu-
gation at 13,000 rpm for 30 min. After the excretion experiment, the cells were washed and
selected in 300 μL HBSS buffer, and then the cells were ultrasonicated. After centrifugation at
13,000 rpm for 30 min, the 200 μL supernatant was obtained and mixed with 100 μL acetoni-
trile containing 25 nM tilianin. The mixture was then centrifuged at 13,000 rpm for 30 min
and prepared for subsequent UHPLC—MS/MS analysis.
The excretion rates, glucuronides intracellular, Glucuronides excretion, the clearance of
efflux transporter (CL) and the fraction of the metabolized dose (fmet) experiments were calcu-
lated as described in detail previously [17].
MTT assay
The cytotoxicity of diosmetin and chrysoeriol on A549 and HepG2 cells was measured using
the MTT assay. Cells were seeded at a density of 3 × 103/well in 96-well plates and grown over-
night, and then the medium was replaced with a fresh medium containing substrates (5 μM
Ko143, 10 μM Ko143, 10 μM diosmetin or chrysoeriol contained or not contained 5 μM
Ko143 or 10 μM Ko143). The cells were further incubated at 37˚C with 5% CO2 for 48 h. The
culture medium was removed and replaced with 100 μL 0.5 mg/mL 3-(4, 5-dimethylthiozol-
2-yl)-3, 5-dipheryl tetrazolium bromide (MTT) solution and incubated at 37˚C for 4 h. The
supernatants were discarded, and 150 μL of dimethyl sulfoxide was added to each well. The
plates were shaken for 10 min at room temperature, and then, the stained formazan product
was determined at 570 nm with a multimode plate reader.
UHPLC—MS/MS system
Quantification of the analytes was carried out on an Agilent 1290 UHPLC system coupled to
Agilent 6490 triple quadrupole MS/MS. The conditions were as follows: column, Acquity
UPLC HSS T3, 1.8 μm, 2.1 mm × 100 mm (Waters, USA); mobile phase A, 100% aqueous
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Glucuronidation and Transportation of Diosmetin and Chrysoeriol
buffer (0.1%, v/v formic acid, pH 2.5); mobile phase B, 100% acetonitrile; flow rate, 0.3 mL/
min; gradient for diosmetin and its metabolites, 0–1 min, 25–25% B, 1–3.5 min, 25–33% B,
3.5–5 min, 33–90% B, 5–6 min, 90–90% B, 6–7 min, 90–21% B; gradient for chrysoeriol and its
metabolites, 0–5 min, 21–21% B, 5–7 min, 21–90% B, 7–8 min, 90–90% B, 8–9 min, 90–21% B;
and injection volume, 2 μL.
The mass spectrometric detection in this study was performed using an ESI source in posi-
tive ionization modes, with capillary voltage set at 3.0 kV, sheath gas and desolvation tempera-
tures set at 300 and 250˚C, respectively. The sheath gas and desolvation flows were 11 and 14
L/min, respectively. The nozzle voltage was set at 1.5 kV. Acquisition was performed in a mul-
tiple-reaction monitoring (MRM) mode at m/z transitions of 447.41!285.20 for tilianin;
477.00!301.00 for Dio-7-G, Dio-30-G, Chr-7-G, and Chr-40-G; and 301.27!286.10 for dios-
metin and chrysoeriol in the positive ESI mode. Collision energies were 20 V for tilianin, 24 V
for diosmetin or chrysoeriol, and 32 V for diosmetin glucuronides or chrysoeriol glucuronides,
respectively.
Statistical Analysis
One-way ANOVA with or without Tukey—Kramer multiple comparison (post hoc) tests were
used to evaluate statistical difference. Data were expressed as mean standard deviation (S.D)
(n = 3), and the level of significance was set at p < 0.05 (“Ô or “#”), p < 0.01 (“ÃÔ or “##”), or
p < 0.001 (“ÃÃÔ or “###”).
Results
Analysis of Diosmetin and Chrysoeriol and their Metabolites by
UHPLC—MS/MS
We already identified the metabolites of diosmetin and chrysoeriol in our previous study [16].
UHPLC—MS/MS analysis showed that two mono-glucuronides were formed in HLMs incu-
bations with diosmetin and chrysoeriol in the presence of UDPGA. The retention times of
diosmetin and its metabolites (Dio-7-G and Dio-30-G) were 5.47, 3.06, and 3.38 min, respec-
tively (Fig 1a). The peak eluting times at 7.35, 5.54, and 6.21 min corresponded to those of
chrysoeriol and its metabolites (Chr-7-G and Chr-40-G) (Fig 1b).
Kinetics of Diosmetin and Chrysoeriol Glucuronidation by HLMs
Diosmetin and chrysoeriol can be metabolized by HLMs into two metabolites, respectively.
Dio-7-G and Dio-3’-G were the metabolites of diosmetin, and the rate of formation Dio-30-G
was much faster than that of Dio-7-G in HLMs within the tested concentration ranges. Hence,
the preferred site for the catalysis of diosmetin glucuronidation was 30-OH rather than 7-OH.
The Eadie—Hofstee plots (Fig 2a and 2b) were used as evidences to state that the glucuronida-
tion of 7-OH glucuronidation exhibited substrate inhibition profiles (Fig 2A), whereas the glu-
curonidation of 30-OH followed the classic Michaelis—Menten kinetics (Fig 2B). Chr-7-G and
Chr-40-G were the metabolites of chrysoeriol, and the rate of formation of Chr-40-G was much
faster than that of Chr-7-G in the HLMs within the tested concentration ranges. Therefore, the
preferred site for the catalysis of chrysoeriol glucuronidation was 40-OH rather than 7-OH.
The Eadie—Hofstee plots (Fig 2c and 2d) were used as evidences to state that the glucuronida-
tion of 7-OH glucuronidation exhibited the classic Michaelis—Menten kinetics (Fig 2C),
whereas 40-OH followed substrate inhibition profiles (Fig 2D).
The kinetic parameters of the glucuronidation of diosmetin and chrysoeriol by HLMs are
shown in Table 1. Dio-30-G in the HLMs exhibited a much lower Km value (0.19 μM for Dio-
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Glucuronidation and Transportation of Diosmetin and Chrysoeriol
Fig 1. Analysis of diosmetin, chrysoeriol, and their metabolites by UHPLC—MS/MS. Fig 1a and 1b show the
chromatogram of the incubation samples of diosmetin and chrysoeriol by HLMs, respectively.
doi:10.1371/journal.pone.0166239.g001
30-G, 4.66 μM for Dio-7-G). The CLint of diosmetin in the HLMs through 30-OH was about 48
times that of the 7-OH pathway (6.25 mL/min/mg for Dio-30-G, 0.13 mL/min/mg for Dio-
7-G). On the other hand, the HLMs exhibited a much lower Km of Chr-40-G than of Chr-7-G
(0.15 μM for Chr-40-G, 3.83 μM for Chr-7-G). As such, the CLint of chrysoeriol in the HLMs
through 40-OH was about 32 times that of the 7-OH pathway (1.27 mL/min/mg for Chr-40-G,
0.04 mL/min/mg for Chr-7-G).
Main UGTs Responsible for the Glucuronidation of Diosmetin and
Chrysoeriol In Vitro
We used UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10,
UGT2B4, UGT2B7, UGT2B15, UGT2B17 to explore which UGT isoform was the main iso-
form in charge of diosmetin or chrysoeriol UGT metabolism (Fig 3). The results showed that
the isoforms that produced the most rapid glucuronidation rates of Dio-7-G (0.21 0.007
nmol/mg/min to 0.85 0.03 nmol/mg/min), Dio-30-G (9.36 0.23 nmol/mg/min to
12.38 0.20 nmol/mg/min), Chr-7-G (0.44 0.01 nmol/mg/min to 0.73 0.01 nmol/mg/min),
Chr-40-G (0.75 0.09 nmol/mg/min to 2.37 0.08 nmol/mg/min) were UGT1A6, UGT1A9,
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Glucuronidation and Transportation of Diosmetin and Chrysoeriol
Fig 2. Kinetics of diosmetin and chrysoeriol glucuronidation by HLMs. The curves were estimated on the
basis of fitted parameters generated using the Michaelis-Menten kinetics (B, C) or substrate inhibition (A, D) in
HLMs. The Eadie—Hofstee plots are shown in right panal (a-d). Each data point corresponds to the average of
three determinations with error bars representing S.D.
doi:10.1371/journal.pone.0166239.g002
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Glucuronidation and Transportation of Diosmetin and Chrysoeriol
Table 1. Kinetic parameters of diosmetin and chrysoeriol glucuronidation by HLMs.
m (μM) Vmax
K
CLint
(mL/min/mg)
0.13
nmol/mg/min
0.61±0.05
1.16±0.03
0.15±0.01
0.20±0.01
diosmetin
Dio-7-G
Dio-30-G
Chr-7-G
Chr-40-G
4.66±0.72
0.19±0.02
3.83±0.55
0.15±0.03
6.25
chrysoeriol
0.04
1.27
doi:10.1371/journal.pone.0166239.t001
UGT1A9 and UGT1A10, respectively. UGT1A3, UGT1A9, UGT1A10 made a prominent con-
tribution to forming Dio-7-G; UGT1A7 made a prominent contribution to forming Dio-30-G;
UGT1A3 and UGT1A8 made a prominent contribution to the formation of Chr-7-G;
UGT1A1 and UGT1A8 made a prominent contribution to the formation of Chr-40-G. UGT2B
family and UGT1A4 had no contribution to diosmetin or chrysoeriol UGT metabolism.
Effects of Chemical Inhibitor on Diosmetin and Chrysoeriol Metabolism
We determined the inhibitory effects of bilirubin (UGT1A1 inhibitor), phenylbutazone
(UGT1A6 inhibitor), and carvacrol (UGT1A9 inhibitor) on the glucuronidation of diosmetin
(Fig 4a1 and 4a2) and chrysoeriol (Fig 4b1 and 4b2) in the HLMs, UGT1A1, UGT1A6 and
UGT1A9. As we can see in Fig 4, the phenylbutazone, calvacrol, calvacrol and bilirubin can
significantly decrease the formation of Dio-7-G, Dio-3’-G, Chr-7-G and Chr-4’-G compared
with control, respectively. And these chemical inhibitors can dose-dependently inhibited the
formation of Dio-7-G, Dio-3’-G, Chr-7-G and Chr-4’-G, which further proved that UGT1A6,
UGT1A9, UGT1A9 and UGT1A1 respectively were the main UGTs involved in the formation
of Dio-7-G, Dio-3’-G, Chr-7-G and Chr-4’-G in HLMs.
Kinetics of Diosmetin and Chrysoeriol Glucuronidation by Human
Expressed UGT enzymes
According to above results, we used human expressed UGT enzymes to incubate different con-
centration diosmetin and chrysoeriol and determined their glucuronidation rates. We used
UGT1A6 and UGT1A9 to incubate diosmetin to further confirm if they were the main isoform
of forming Dio-7-G and Dio-30-G, respectively; and we used UGT1A9 and UGT1A1 to incu-
bate chrysoeriol to further confirm if they were the main isoform of forming Chr-7-G and
Chr-40-G, respectively. Within the tested concentration ranges, The Eadie—Hofstee plots (Fig
5a and 5d) were used as evidences to state that both UGT1A6- and UGT1A1-mediated the for-
mation of Dio-7-G (Fig 5A) and Chr-40-G (Fig 5D) exhibited substrate inhibition kinetic char-
acteristics. And the Eadie—Hofstee plots (Fig 5b and 5c) were used as evidences to state that
both UGT1A9-mediated formation of Dio-30-G (Fig 5B) and Chr-7-G (Fig 5C) exhibited clas-
sic Michaelis-Menten kinetic characteristics.
The kinetic parameters of the diosmetin and chrysoeriol glucuronidation catalyzed by
human expressed UGT enzymes are shown in Table 2. These data accompanied the absence of
large variation in Km value of the glucuronides except for Dio-7-G (3.02 μM for Dio-7-G in
UGT1A6, 0.45 μM for Dio-30-G in UGT1A9, 0.26 μM for Chr-7-G in UGT1A9, and 0.28 μM
for Chr-40-G in UGT1A1). The intrinsic clearance (CLint; Vmax/Km) of Dio-7-G, Dio-30-G,
Chr-7-G, and Chr-40-G selectively in UGT1A6, UGT1A9, UGT1A9, and UGT1A1 were higher
than that in HLMs.
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Glucuronidation and Transportation of Diosmetin and Chrysoeriol
Fig 3. Glucuronidation of diosmetin and chrysoeriol by human expressed UGT isoforms. Experiments
were conducted at concentrations 1.25, 2.5, and 10 μM. The concentration of each human expressed UGT
isoform was 0.0053 mg/mL. The experiments were conducted at 37˚C for 30 min, and the amount of
glucuronides formed were measured by UHPLC—MS/MS. The rates of glucuronidation were calculated as
nmol/mg/min. Each column corresponds to the average of three determinations with error bars representing
the S.D.
doi:10.1371/journal.pone.0166239.g003
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Glucuronidation and Transportation of Diosmetin and Chrysoeriol
Fig 4. Effects of chemical inhibitors on diosmetin and metabolism and UGT1A9- and UGT1A1-mediated chrysoeriol metabolism in HLMs. Fig 4
A shows the inhibitory effects of phenylbutazone on the 7-O-glucuronide of diosmetin in HLMs and UGT1A6. Fig 4 B displays the inhibitory effects of
carvacrol on the 30-O-glucuronide of diosmetin in HLMs and UGT1A9. Fig 4 C presents the inhibitory effects of carvacrol on the 7-O-glucuronide of
chrysoeriol in HLMs and UGT1A9. Fig 4 D shows the inhibitory effects of bilirubin on the 40-O-glucuronides of chrysoeriol in HLMs and UGT1A1. Each
column corresponds to the average of three determinations with error bars representing the S.D. The “*” symbol means a statistically significant difference
compared with control at p < 0.05; “**” means p < 0.01; “***” means p < 0.001.
doi:10.1371/journal.pone.0166239.g004
Effects of Diosmetin and Chrysoeriol Concentrations on Glucuronide
Excretion
Six concentrations of diosmetin and chrysoeriol were chosen to explore the effects of diosme-
tin and chrysoeriol concentrations on the excretion, intracellular amount, CL, and fmet in Hela
and HeLa—UGT1A9 cells (Fig 6). Because it did not find the glucuronided metabolites of
diosmetin or chrysoeriol in HeLa cells, we mainly determined the excretion, intracellular
amount, CL, and fmet in HeLa—UGT1A9 cells. (Fig 6). The excretion rates of Dio-7-G reached
a peak at 10 μM and Dio-30-G reached a peak at 2.5 μM (Fig 6a1). The excretion rates of Chr-
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Glucuronidation and Transportation of Diosmetin and Chrysoeriol
Fig 5. Kinetics of diosmetin and chrysoeriol glucuronidation by human expressed UGT enzymes. The
curves are estimated on the basis of fitted parameters generated using the substrate inhibition (A, D) or
Michaelis-Menten kinetics (B, C) in UGT1A6, UGT1A1, UGT1A9, and UGT1A9, respectively. The Eadie—
Hofstee plots are shown in the right panal (a-d). Each data point corresponds to the average of three
determinations with error bars representing the S.D.
doi:10.1371/journal.pone.0166239.g005
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Glucuronidation and Transportation of Diosmetin and Chrysoeriol
Table 2. Kinetic parameters of diosmetin and chrysoeriol glucuronidation by human expressed UGT enzymes.
K
m (μM)
Vmax
CLint
mL/mg/min
0.30
nmol/mg/min
0.92±0.06
4.27±0.13
0.12±0.01
0.63±0.06
diosmetin
Dio-7-G (UGT1A6)
Dio-30-G (UGT1A9)
Chr-7-G (UGT1A9)
Chr-40-G (UGT1A1)
3.02±0.59
0.45±0.05
0.26±0.04
0.28±0.05
9.52
chrysoeriol
0.48
2.26
doi:10.1371/journal.pone.0166239.t002
7-G and Chr-40-G reached a peak at 2.5 μM, (Fig 6a2). Intracellular glucuronide levels dose-
dependently increased with diosmetin or chrysoeriol loading concentrations (Fig 6b1 and
6b2). CL (Fig 6c1 and 6c2) dose-dependently decreased with increasing diosmetin or chrysoer-
iol concentration. And fmet (Fig 6d1 and 6d2) dose-dependently decreased with increasing
diosmetin or chrysoeriol concentration except Chr-40-G. The fmet of Chr-40-G reached a peak
at 2.5 μM chrysoeriol.
Effect of Ko143 on Glucuronides Excretion
To estimate the effect of BCRP on the excretion of glucuronides in HeLa—UGT1A9 cells, the
Ko143 was adopted (Fig 7). Due to at the excretion rate of diosmetin glucuronides or chrysoer-
iol glucuronides reached a peak at 2.5 μM substrate concentration, the concentration of sub-
strate were set at 2.5 μM. Co-incubation of 5 μM or 10 μM Ko143 with diosmetin or
chrysoeriol led to the excretion rates of diosmetin or chrysoeriol glucuronides decreased and
their intracellular level significantly increased. Ko143 (5 μM) inhibited Dio-7-G and Dio-30-G
excretion by 19% and 16%, respectively; and Ko143 (10 μM) decreased the excretion by 40%
and 29%, respectively (Fig 7a1 and 7a2). Moreover, 5 μM Ko143 inhibited Chr-7-G and Chr-
40-G excretion by 14% and 19%, respectively; and 10 μM Ko143 decreased both excretions by
43% (Fig 7a3 and 7a4). The effects of Ko143 on the total intracellular amount of glucuronides
are displayed in Fig 7B. At 2.5 μM diosmetin, the total amount of intracellular Dio-7-G and
Dio-30-G was increased by 1.1- and 1.3-fold, respectively, in the presence of 5 μM Ko143 and
by 1.5- and 1.8-fold, respectively, in the presence of 10 μM Ko143 compared with the control
(Fig 7b1). At 2.5 μM chrysoeriol, the total amount of intracellular Chr-7-G and Chr-40-G both
increased by 1.3-fold in the presence of 5 μM Ko143 and by 1.7- and 1.8-fold, respectively, in
the presence of 10 μM Ko143 compared with the control (Fig 7b2). Ko143 inhibited the CL of
these four glucuronides (33% to 65%) (Fig 7C). The results of fmet (Fig 7D) indicated that the
effect of Ko143 on diosmetin or chrysoeriol glucuronidation were small or insignificant. All
the results indicated that the BCRP played a crucial role in excretion diosmetin glucuronides
and chrysoeriol glucuronides.
The effects of diosmetin and chrysoeriol on cell viability in A549 and
HepG2 cells with untreated and treated Ko143
We found that in A549 cells, 10 μM chrysoeriol with 5 μM or 10 μM Ko143 respectively
decreased the A549 cell viability by 12% and 18% compared with the group that substrate was
10μM chrysoeriol, but the cytotoxicity of diosmetin had little change when treated Ko143.
And in HepG2 cells, 10 μM diosmetin with 5 μM or 10 μM Ko143 respectively decreased the
A549 cell viability by 12% and 13% compared with the group that substrate was 10 μM diosme-
tin, but the cytotoxicity of chrysoeriol had little change when treated Ko143 (Fig 8).
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Glucuronidation and Transportation of Diosmetin and Chrysoeriol
Fig 6. Effect of substrate concentrations on excretion rates (a), intracellular amounts (b), CL (c), and
met (d) of Dio-7-G, Dio-30-G, Chr-7-G, and Chr-40-G. Three samples (500 μL) were obtained at 15, 30, and
f
60 min and replaced with fresh loading solution (500 μL) that contains diosmetin or chrysoeriol. The excretion
rates of glucuronides were calculated from the slope of the amount-versus-time curves. The intracellular
amounts of the glucuronides were determined at the end of the excretion experiments after the cells were
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Glucuronidation and Transportation of Diosmetin and Chrysoeriol
washed twice with ice-cold HBSS. Each column corresponds to the average of three determinations with error
bars representing the S.D. The “*” (for Dio-7-G or Chr-7-G) or “#” (for Dio-30-G or Chr-40-G) symbol means a
statistically significant difference between-group, at p < 0.05; “**” or “##” means p < 0.01; “***” or “###” means
p < 0.001.
doi:10.1371/journal.pone.0166239.g006
Discussion
Luteolin is widely found in our diet, such as carrots, peppers, celery, olive oil and peppermint
[17]. It was reported that luteolin could be metabolized to diosmetin and chrysoeriol, which
were the methylation metabolites. And the diosmetin and chrysoeriol could be metabolized in
rats by Ugts [16]. Therefore, it was speculated that glucuronides of diosmetin and chrysoeriol
would be formed in humans. The objective of this study was to the metabolism characteristic of
diosmetin and chrysoeriol. The exposure of the glucuronides of diosmetin and chrysoeriol
could influence the pharmacological action of luteolin. Hence, the metabolic elucidation of
diosmetin and chrysoeriol is important to understand the pharmacokinetic characteristics and
biological effects of luteolin in vivo. Diosmetin and chrysoeriol, which also distributed in many
plant products, are pharmacologically valuable in many diseases, such as osteoporosis [4]. This
study for the first time investigated the regioselectivity of the glucuronidation of diosmetin and
chrysoeriol by kinetic profiling and identified the major enzymes responsible for the glucuroni-
dation of the two compounds. In addition, this study also showed the effect of the BCRP—
UGT1A9 interplay on diosmetin and chrysoeriol glucuronidation in vivo.
The metabolic activities of the HLMs toward diosmetin and chrysoeriol were determined
by kinetic profiling (Fig 2). These activities included the metabolic rates at various substrate
concentrations. The CLint value, independent of substrate concentration, measures the cata-
lytic efficiency of functional enzymes. The determination of regioselectivity was based on the
CLint derived from kinetic profiling over a wide range of substrate concentrations. The eluci-
dation of regioselectivity by kinetic determination was superior to that by rate determination
at high substrate concentrations [31]. In addition, the use of CLint as indicator of UGT activity
was more advantageous than the use of reaction rates because CLint is more relevant in predict-
ing hepatic clearance in vivo compared with other kinetic parameters [32,33]. In HLMs, the
CLint value of diosmetin through 30-OH was much higher than that of the 7-OH, and the CLint
value of chrysoeriol through 40-OH was about 32 times than that of the 7-OH pathway. For
another, the Km values of diosmetin and chrysoeriol glucuronides were lower than a series of
7-OH, 3’-OH or 4’-OH flavonoids, such as acacetin glucuronide (Km was 14.465 μM)[34] and
genistein glucuronides (Km was 15.1 μM)[35] (Table 1).
On the other hand, human expressed UGTs and chemical inhibition assays were used to
determine the major UGTs involved in the glucuronidation metabolism of diosmetin and
chrysoeriol. Among the 12 expressed UGTs, only the enzymes from the UGT1A family except
UGT1A4 showed glucuronidation activity on diosmetin and chrysoeriol, and the UGT2B fam-
ily offered no contribution to their UGT metabolism. According to the expressed UGT assay,
the main UGT isoforms for Dio-7-G, Dio-30-G, Chr-7-G, and Chr-40-G formation were
UGT1A6, UGT1A9, UGT1A9 and UGT1A10, respectively (Fig 3). Because the expression of
individual UGTs at the mRNA level in the liver are different (UGT1A1 (7%), UGT1A6 (5%),
UGT1A9 (6%))[36], and the UGT isoform showing the highest activity could not be the main
contributor in HLMs. Therefore, the inhibition assay was further performed to determine the
role of the UGT isoform in the metabolism of diosmetin and chrysoeriol. In addition, it was
reported that UGT1A8 and 1A10, which were involved in the glucuronidation of diosmetin
and chrysoeriol, were not present in the liver [37]. And the objective of this research was to
study the hepatic clearance of diosmetin and chrysoeriol. Thus, the effect of UGT1A8 and
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Glucuronidation and Transportation of Diosmetin and Chrysoeriol
Fig 7. Effect of BCRP-specific inhibitor Ko143 on the excretion rate of glucuronides (Dio-7-G, Dio-30-
G, Chr-7-G, and Chr-40-G) (a), total intracellular glucuronide amounts (Dio-7-G, Dio-30-G, Chr-7-G, and
Chr-40-G) (b), CL (c), and fmet (d). Engineered HeLa cells stably overexpressing UGT1A9 grown on six-well
plates (1 × 105 cells/well) were treated with 2.5 μM of diosmetin or chrysoeriol in the absence or presence of
Ko143 at 5 or 10 μM. Three samples (500 μL) were obtained at 20, 40, and 60 min and replaced with fresh
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Glucuronidation and Transportation of Diosmetin and Chrysoeriol
loading solution (500 μL) containing 2.5 μM diosmetin or chrysoeriol. Each data point (panel a) and each
column (panel b-d) corresponds to the average of three determinations with error bars representing the S.D.
The “*” (for Dio-7-G or Chr-7-G) or “#” (for Dio-30-G or Chr-40-G) symbol means a statistically significant
difference between-group at p < 0.05; “**” or “##” means p < 0.01; “***” or “###” means p < 0.001.
doi:10.1371/journal.pone.0166239.g007
1A10 was not further consideration. Therefore, UGT1A1, which was the second main isoform
for Chr-40-G formation, and UGT1A6, UGT1A9 were used to the further inhibition assay, and
these three isoforms are highly expressed in liver [37]. Bilirubin (UGT1A1 inhibitor), phenyl-
butazone (UGT1A6 inhibitor) and carvacrol (UGT1A9 inhibitor) were used in this study. All
the concentrations of diosmetin and chrysoeriol adopted in this study were based on the Km
value of glucuronides. The results showed that bilirubin, phenylbutazone, and carvacrol inhib-
ited the formation of the glucuronides in UGT enzymes and HLMs in a dose-dependent man-
ner (Fig 4). Thus, the results suggested that UGT1A6, UGT1A9, UGT1A9, and UGT1A1 were
the main expressed UGTs responsible for the formation of Dio-7-G, Dio-30-G, Chr-7-G, and
Chr-40-G, respectively. The inhibition kinetics of these three inhibitors have been previously
described to be substrate dependent, but the mechanism of inhibition of drug-metabolizing
enzymes remains unclear [29,38]. Thus, the mechanism of inhibition of these inhibitors
requires additional studies, and in vitro data on the inhibition should be interpreted with cau-
tion. To further elucidate the characteristics of the glucuronidation metabolism of diosmetin
and chrysoeriol, their rates of glucuronidation metabolism by UGT1A1, UGT1A6, and
UGT1A9 were determined at different substrate concentrations (Fig 5). The results of compa-
rable kinetic profiles of the expressed UGTs further explained the conclusion in chemical inhi-
bition study (Table 2).
In this study, UGT1A9 was the main expressed UGTs responsible for the formation of Dio-
30-G and Chr-7-G, and Dio-7-G and Chr-40-G also can be formed by UGT1A9. Therefore, the
Fig 8. The effects of diosmetin and chrysoeriol on cell viability in A549 and HepG2 cells with untreated and treated Ko143. 5 μM Ko143, 10 μM
Ko143, 10 μM diosmetin, 10 μM diosmetin with 5 μM Ko143 or 10 μM Ko143, 10μM chrysoeriol, 10μM chrysoeriol with 5 μM Ko143 or 10 μM Ko143 were
used as substrates to determine the effects of diosmetin and chrysoeriol on cell viability in A549 and HepG2 cells with untreated and treated Ko143. Each
column corresponds to the average of three determinations with error bars representing the S.D. The “*” symbol means a statistically significant difference
compared with control at p < 0.05; “**” means p < 0.01; “***” means p < 0.001).
doi:10.1371/journal.pone.0166239.g008
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Glucuronidation and Transportation of Diosmetin and Chrysoeriol
HeLa—UGT1A9 cells, which were used to study how the interplay between UGT1A9 and
BCRP affects drug disposition [21], were utilized to further elaborate diosmetin and chrysoer-
iol metabolism in terms of glucuronide production. This work delineated the interplay
between UGT1A9 and BCRP in diosmetin and chrysoeriol metabolism and disposition at the
kinetic level. The excretion rates of Dio-7-G in HeLa—UGT1A9 peaked at 10 μM, and those of
Dio-30-G, Chr-7-G, and Chr-40-G rapidly peaked at 2.5 μM (Fig 6). The glucuronide formation
did not show substrate inhibition kinetics; hence, we speculated that the decrease in the excre-
tion rates of the glucuronides at high diosmetin or chrysoeriol concentration was most likely
caused by BCRP inhibition by diosmetin or chrysoeriol. Indeed, several flavonoids (e.g., aya-
nin and retusin) have been reported to inhibit BCRP encoded by the ABCG2 gene with poten-
cies only slightly lower than that of Ko143. Structural features found to contribute positively to
BCRP inhibition included a hydroxyl group in position 5 and a double bond between position
2 and 3 [39], which were also found in the structures of diosmetin and chrysoeriol. Therefore,
BCRP inhibition by diosmetin or chrysoeriol was believed to account for the decreased excre-
tion rate of glucuronides in the HeLa—UGT1A9 cells observed at high diosmetin or chrysoer-
iol concentrations.
The role of BCRP in the excretion was studied using Ko143, a specific BCRP inhibitor that
does not affect UGT activities as a general rule. Ko143 (5 μM or 10 μM) significantly inhibited
the BCRP-mediated excretion of the glucuronides of diosmetin and chrysoeriol. Ko143 also
significantly increased the accumulation of intracellular glucuronides at 2.5 μM (Fig 7). Impor-
tantly, Ko143 inhibited glucuronide CL and fmet at 2.5 μM diosmetin or chrysoeriol, suggesting
that BCRP-mediated excretion could be the predominant pathway for diosmetin and chry-
soeriol disposition.
According to the results of the effect of Ko143 on glucuronides excretion, we found that the
total intracellular amount of glucuronides was increases after treated Ko143. But the effects of
increased intracellular glucuronides in cells were not clear. Therefore, we used these A549 and
HepG2 tumor cell lines to demonstrate the effects of diosmetin and chrysoeriol on cell viability
with untreated and treated Ko143 inhibitor (Fig 8). Both A549 and HepG2 cells express BCRP
[40,41]. A549 cells express UGT1A1, UGT1A3 and UGT2B7 [42], and HepG2 cells expresse
UGT1A1, UGT1A6 and UGT1A9 [43]. According to the present results, we found that the
increased total intracellular glucuronides could increase the cytotoxicity, and diosmetin and
chrysoeriol had different effects on different cancer cells, possibly due to different isoform of
UGTs in the different cancer cells.
In conclusion, this study first demonstrated that the diosmetin and chrysoeriol could be
metabolited into Dio-7-G, Dio-30-G, Chr-7-G and Chr-40-G by human in vitro. All the find-
ings suggested that the oral bioavailability of luteolin, diosmetin and chrysoeriol would be
greatly limited by first-pass glucuronidation in the liver. In addition, this work established that
BCRP-mediated excretion was the predominant pathway for diosmetin and chrysoeriol dispo-
sition, which could help increase the understanding and prediction of BCRP-mediated glucu-
ronide CL in vivo. Our studies contribute to improving the understanding on the
pharmacokinetic characteristics and biological effects of luteolin in vivo.
Author Contributions
Conceptualization: XW ZL.
Data curation: XZ QC JS LS LL.
Formal analysis: XZ HJ JS LZ LL.
Funding acquisition: XW ZL.
PLOS ONE | DOI:10.1371/journal.pone.0166239 November 10, 2016
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Glucuronidation and Transportation of Diosmetin and Chrysoeriol
Investigation: XZ QC LZ XW ZL.
Methodology: XZ QC HJ JS LZ.
Project administration: XW ZL.
Resources: LZ LL XW ZL.
Software: XZ JS LZ LL MZ.
Supervision: XW ZL.
Validation: QC JS LZ LL MZ.
Visualization: LW FL LZ LL.
Writing – original draft: XZ JS LZ ZL.
Writing – review & editing: LZ LL XW ZL.
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