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Sulfonation Disposition of Acacetin: In Vitro and In Vivo Qisong Zhang, Lijun Zhu, Xia Gong, Yanjiao Ruan, Jia Yu, Huangyu Jiang, Ying Wang, Xiaoxiao Qi, Linlin Lu, and Zhong Qiu Liu J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 25 May 2017 Downloaded from http://pubs.acs.org on May 25, 2017
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Journal of Agricultural and Food Chemistry
Sulfonation Disposition of Acacetin: In Vitro and In Vivo
Qisong Zhang,1, 2 Lijun Zhu,2 Xia Gong,2 Yanjiao Ruan,2 Jia yu,2 Huangyu Jiang,2 Ying Wang,2 XiaoXiao Qi,2 Linlin Lu,2* and Zhongqiu Liu1, 2* 1. Department of Pharmaceutics, School of Pharmaceutical Sciences, Southern Medical University, GuangZhou, GuangDong, 510515, China. 2. International Institute for Translational Chinese Medicine, GuangZhou University of Chinese Medicine, GuangZhou, GuangDong, 510006, China.
Corresponding Author Prof. Dr. ZhongQiu Liu, Email:
[email protected] and
[email protected] Phone: +8620-39358061
Fax: +8620-39358071
Dr. LinLin Lu, Email:
[email protected] Phone: +8620-39357902
Fax: +8620-39358071
1
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ABSTRACT: Acacetin, an important component of acacia honey, exerts extensive
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therapeutic effects on many cancers. However, sulfonation disposition of acacetin has
3
rarely been reported. Therefore, this study aims to investigate the sulfonation
4
disposition of acacetin systematically. Results showed that acacetin-7-sulfate was the
5
main metabolite mediated primarily by sulfotransferases (SULT) 1A1. Dog liver S9
6
presented the highest formation rate of acacetin-7-sulfate. Compared with that in
7
wild-type Friend Virus B (FVB) mice, plasma exposure of acacetin-7-sulfate
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decreased significantly in multiple drug resistance protein 1 knockout (Mrp1−/−) mice,
9
while increased evidently in breast cancer resistance protein knockout (Bcrp−/−) mice.
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In Caco-2 monolayers, efflux and clearance of acacetin-7-sulfate reduced distinctly by
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the BCRP inhibitor Ko143 in apical side and by the MRP1 inhibitor MK571 in
12
basolateral side. In conclusion, acacetin sulfonation was mediated mostly by
13
SULT1A1. Acacetin-7-sulfate was transported mainly by BCRP and MRP1. Hence,
14
SULT1A1, BCRP and MRP1 were responsible for acacetin-7-sulfate exposure in vivo.
15 16
Keywords: Acacetin; Sulfonation; Metabolism; Transport; Pharmacokinetics
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INTRODUCTION
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Honey has been flavorful for many decades because of its high nutritional value
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and contribution to human health 1. Acacia honey, produced by bees in acacia flowers,
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is widely consumed all year round in many countries and is the most popular in
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Malaysia 2. Acacetin, an important flavone in acacia honey, is one of the main
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flavonoids accounting for 28.83-113.06 mg/kg of whole honey composition
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Acacetin also presents numerous pharmacological effects, such as anti-peroxidative,
8
anti-inflammatory, anti-plasmodia
9
cancer, prostate cancer and stomach cancer
4-8
2, 3
.
and therapeutic effects on breast cancer, liver 4, 7, 9, 10
. Moreover, Gui-Rong et al.
10
reported that oral acacetin was a promising atrium-selective agent for treatment of
11
atrial fibrillation. However, most flavonoids could undergo extensive phase II
12
metabolism mediated by UDP-glucuronosyltransferases (UGT) and sulfotransferases
13
(SULT) in vivo. A clear metabolic study of acacetin may be helpful for the daily use of
14
acacia honey.
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Glucuronidation for acacetin has been extensively investigated in our laboratory 11, 12
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through studies in rats and recombinant human UGTs
17
sulfonation has been rarely reported. Sulfonation metabolism is catalyzed by members
18
of the SULT family 13, such as SULT1, SULT2, SULT4 and SULT6. SULTs perform
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various important functions with distinct tissue distribution for each individual
20
using the universal sulfonyl donor molecule 3'-phosphoadenosine-5'-phosphosulfate
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(PAPS) to form sulfate
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cellular reactions that modify lots of xenobiotics and endogenous substances
14, 15
. However, acacetin
14
by
. Sulfonation is one of the most abundant and important
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regulating important biological processes including blood clotting, formation of
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connective tissues, and functionality of secreted proteins, hormones and signaling
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molecules 16.
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The activation and inactivation of numerous xenobiotics and endogenous
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compounds occur by sulfonation pathway, which may change the physiological
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function of body. For instance, the sulfonation of certain drugs may enhance
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therapeutic activity. Minoxidil is a drug for antihypertensive and hair growth
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stimulating, and tamoxifen is a common drug for treating breast cancer, both of their
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sulfate metabolite are responsible for their biological activities
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sulfonation is the most abundant post-translational modification of tyrosine residues
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implicated in numerous physiological and pathological processes
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acts as a determinant of protein–protein interactions, which are involved in leukocyte
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adhesion, hemostasis and receptor-mediated signaling
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essential for proper blood clotting in response to vessel injuries and binding of
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chemokines to their receptors CCR5 and CXCR4
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showed that individual differences in various genes of sulfonation pathway may
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contribute to carcinogenesis and patient survival. For example, polymorphisms in
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SULT1E1, which involves in estrogen sulfonation, are related to the survival of
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patients with estrogen-dependent cancers. Hirata et al. and Rebbeck et al. found that
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polymorphisms in SULT1E1 are associated with high endometrial cancer risks 22, 23.
43 44
20, 21
19
18
17
. Furthermore,
. Tyrosine sulfate
. Tyrosine sulfate is also
. In addition, recent evidences
Furthermore, increasing evidences showed that metabolic enzymes and efflux transporters exert synergistic effects on the disposition of flavonoids in vivo 4
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.
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Previous reports also showed that many flavonoid conjugates are substrates of efflux
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transporters (e.g., p-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and
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multiple drug resistance proteins (MRPs)), which are expressed abundantly in the
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intestine and liver
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determined by sulfotransferases, efflux transporters or both and which efflux
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transporters and sulfotransferases involved in this process remain unknown. Hence,
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elucidation of sulfonation disposition of acacetin in vivo could help gain a further
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systematic understanding of acacetin metabolic and disposition characteristics in vivo.
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To further understand the sulfonation disposition of acacetin, incubation in vitro,
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pharmacokinetics and Caco-2 cell monolayer models were utilized in the present
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study. A sensitive and reliable LC-MS/MS method was developed to determine
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acacetin and its sulfate accurately, directly and simultaneously. The metabolic
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characteristics of species and isoforms were used to study the sulfonation activity of
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different species and ascertain major isoform mediating acacetin sulfonation.
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Pharmacokinetics of knockout mice and wild-type mice provided insights into the
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effect of efflux transporters on the exposure level of acacetin sulfate. The Caco-2 cell
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model combined with selective inhibitors was used as an in vitro approach to confirm
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the role of efflux transporters.
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MATERIALS AND METHODS
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Chemicals and Reagents. Acacetin (≥98%, HPLC grade) was purchased from
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Chengdu Must Biotechnology Co., Ltd. (Chengdu, China). Acacetin-7-sulfate
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(Aca-7-S) was purified from incubation in vitro and identified using electrospray
25
. However, whether the sulfonation disposition of acacetin is
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ionization mass spectrometry and diode array detector (DAD) as previously
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described
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Merck Company (Merck Millipore, USA). Magnesium chloride, dimethyl sulfoxide
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(DMSO), β-cyclodextrins and PAPS were purchased from Sigma-Aldrich (St. Louis,
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MO, USA). Recombinant human SULTs (1A1, 1B1, 1E1, 2A1 and 2B1) were
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purchased from R&D Systems Co., Ltd (Guangzhou, China). Human pooled liver S9
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(150 donors), and pooled liver S9 of mouse, rat, monkey and dog were purchased
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from Gene Co., Ltd (Guangzhou, China). Pooled liver, intestine and colon S9
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fractions (centrifuged tissue homogenate for 20 min at 9000 x g
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contained cytosolic fractions and microsomal fractions
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knockout mice were prepared in our laboratory. All other reagents were typically
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analytical grade and used as received.
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Animals. Wild-type FVB mice were purchased from Vital river Co., Ltd (Beijing,
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China). Bcrp−/−, Mrp1−/− and Mrp2−/− mice were ordered from Shanghai Biomodel
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Organism Science and Technology Development Co., Ltd (Shanghai, China). The
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mice weighted 20~ 30 g, and they were 8~10 weeks old.
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LC-MS/MS Method Development. Agilent 6540 Accurate-Mass quadrupole time
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of flight (Q-TOF) MS System and Agilent 6490 QQQ MS tandem Agilent 1290
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UHPLC System were used for qualification and quantification of acacetin and its
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sulfate, respectively. The LC conditions were as follows: column, ZORBAX SB-C18,
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1.8 μm, 3.0 mm × 100 mm; mobile phase A, 100% aqueous buffer (0.01%, v/v
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formic acid, pH 4); mobile phase B, 100% acetonitrile, flow rate, 0.35 mL/min; and
26
. Formic acid and acetonitrile (HPLC grade) were purchased from US
29
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, which mainly
) of wild-type FVB and
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gradient: 0–1.5 min, 30%B, 1.5–2.5 min, 30%–70% B, 2.5–3.5 min, 70%–100% B,
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3.5–4.5 min, 100%–80% B, 4.5–5.5 min, 80%–30%B.
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The 6490 QQQ MS spectrometer parameters were as follows: fragmentor
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voltage: 380 V; capillary voltage: 3000 V; nozzle voltage: 1500 V; nebulizer
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pressure: 20 psi; sheath gas temperature: 250 °C; and gas temperature: 200 °C.
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Sheath gas flow rate and gas flow rate of 11 and 14 L/min, respectively. Injection
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volume: 5 μL; column temperature: 35 °C. Data acquisition and analysis were
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performed using Agilent Mass hunter software. The observed compound m/z value
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were as follows: Aca-7-S (363.1→282.9); testosterone (289.01→97.2); and acacetin
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(285→242).
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Preparation of Mouse Liver, Small Intestine and Colon S9 Fractions. Male
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wild-type, Bcrp−/−, Mrp1−/− and Mrp2−/− FVB mice (n=10) were kept in an
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environmentally controlled room (temperature: 25 ± 2 °C, humidity: 50 ± 5%, and
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12 h dark-light cycle) for at least three days prior to the experiments. The S9
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fractions were prepared using a procedure published previously with minor
104
modification
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centrifuge tubes at −80 °C. And the S9 fractions protein concentration was
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determined by the Bio-Rad protein assay (Bio-Rad Laboratories, USA) using bovine
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serum albumin as the standard.
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Species-dependent Sulfonation of Acacetin in Liver S9 Fractions of Different
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Species (Human, Monkey, Dog, Rat and Mouse). The sulfonation reaction system
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and incubation procedures were the same as those previous study
30-32
. The liver, intestine and colon S9 fractions of mice were stored in
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. Briefly, the
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reaction mixture (total volume 200 μL) consists of 10 μL enzymes (liver S9 fractions
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of species, 0.025 mg/mL), 5 μL magnesium chloride (1 mM), 5 μL PAPS (0.05 mM),
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178 μL KPI buffer (44.5 mM, pH 7.4) and 2μL acacetin (3, 6, and 24 nM). The S9
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fractions or substrate must be added lastly. The mixture prepared in equal triplicates
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was incubated in a 37 °C shaking water bath (speed=50 rpm) for 20-30 min. The
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metabolic percentage of acacetin was not over 30%. Finally, the reactions were
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stopped by adding a semisystem volume of the testosterone acetonitrile solution. The
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mixture system was vortexed for 3min, and centrifuged at 19375 × g for 30 min,
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4 °C. The supernatant was directly subjected to LC–MS/MS for analysis.
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SULT Isoform-specific Metabolic Characteristics of Acacetin by Recombinant
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Human SULTs (1A1, 1B1, 1E1, 2A1 and 2B1). The sulfonation reaction system and
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incubation procedures were identical to the system for species-dependent sulfonation
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of acacetin. Briefly, the reaction mixture (total volume 200 μL) with acacetin (3, 6,
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and 24 nM) was mediated by SULT isoforms (0.0025 mg/mL). Time for incubation
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was 20-30 min. The metabolic percentage of acacetin was also not over 30%. Lastly,
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the reactions were stopped by the testosterone acetonitrile solution. The mixture
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system was centrifuged at 19375 × g for 30 min, 4 °C, and the supernatant was
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analyzed directly by LC–MS/MS.
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Pharmacokinetic Studies in the Wild-type, Bcrp−/−, Mrp1−/− and Mrp2−/− FVB
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Mice. The FVB mice were used extensively in transgenic research because of its
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defined inbred background (high gene homology and little individual difference),
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superior reproductive performance, and prominent pronuclei, which facilitated 8
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microinjection of genomic material and served as the background model mice of
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Bcrp−/−, Mrp1−/− and Mrp2−/− mice in our study. The male wild-type, Bcrp−/−, Mrp1−/−
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and Mrp2−/− FVB mice were kept in an environmentally controlled room for at least
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1 week prior to the experiments. Animal experiments were performed in accordance
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with the Guide for the Care and Use of Laboratory Animals by the National
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Institutes of Health, and the procedures were approved by the Ethical Committee of
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Guangzhou University of Chinese Medicine (Guangzhou, China). The acacetin
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solution system consisted of 5% DMSO, 5% ethanol and 90% β-cyclodextrin
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aqueous solution (1g β-cyclodextrins: 4 mL 0.9% normal saline) at 0.5 mg/mL. Prior
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to pharmacokinetic experiments, the wild-type, Bcrp−/−, Mrp1−/− and Mrp2−/− FVB
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mice (n=5) were fast for at least 8 h. The acacetin solution was administered orally
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to the mice at a dose of 5 mg/kg. Blood samples (about 30 μL) of wild-type, Bcrp−/−,
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Mrp1−/− and Mrp2−/− FVB mice were collected from 5 mice of the same strain at
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each time point by the tail vein to heparinized tubes at 0, 3, 5, 10, 15, 20, 30, 60, 180,
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300, 420, 540, 720 and 1440 min, and then centrifuged at 11481×g for 8 min at 4 °C.
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Afterward the plasma supernatant were collected and stored at −20 °C until analysis.
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Enzyme Kinetic Studies of Acacetin Sulfonation by Mouse Liver, Small
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Intestine and Colon S9 Fractions. The sulfonation reaction system and incubation
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procedures were also identical to the system of species-dependent acacetin
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sulfonation. Briefly, the reaction mixture (total volume 200 μL) with acacetin
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(0.005-20 μM) was mediated by S9 fractions (0.025-0.05 mg/mL) from mouse. Time
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for incubation was 15-30 min. Formation rates of acacetin sulfonation were 9
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determined by the amounts of metabolite formed per mg protein per min
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(pmol/mg/min). The data were obtained from triplicate reactions. Kinetic parameters
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estimated by fitting the initial rate to the rate equations and by subsequent nonlinear
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least-squares regression were obtained according to the profile of Eadie-Hofstee
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plots
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Aca-7-S at respective substrate concentrations (C) were suitable for the standard
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Michaelis-Menten equation:
34, 35
. When the Eadie-Hofstee plot was linear, the formation rates (V) of
𝑉=
𝑉𝑚𝑎𝑥 × 𝐶 𝐾𝑚 + 𝐶
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Where Km is the Michaelis-Menten equation constant, and Vmax is the maximum rate
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of sulfate formation. When Eadie-Hofstee plots displayed characteristic profiles of
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atypical kinetics 34, 36, data from these atypical profiles were suitable for Eq (1) and (2)
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by using the ADAPT II program. To confirm the best-fit model, model candidates
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were assessed using the Akaike's information criterion (AIC) and R2 value
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Therefore, using this minimum AIC estimation, a negative AIC value is considered a
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better indication of data compared with that of a data set with a positive AIC value.
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Reaction rate =
[𝑉𝑚𝑎𝑥 −0 +𝑉𝑚𝑎𝑥 −𝑑 (1−𝑒 −𝐶𝑅 )]×𝐶 𝐾𝑚 +𝐶
34
.
(1)
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Where Vmax-0 is the maximal intrinsic enzyme activity, Vmax-d is the maximal inducible
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enzyme activity, R is the rate of enzyme activity induction, C is the concentration of
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substrate, and Km is the substrate concentration to achieve 50% of (Vmax-0 + Vmax-d).
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Substrate inhibition kinetics:
174 175
Reaction rate =
𝑉𝑚𝑎𝑥 1 1+(𝐾𝑚 1 /𝐶）+(𝐶/𝐾𝑠𝑖 )
(2)
Where Vmax1 is the maximum enzyme activity, C is the substrate concentration, Km1 is 10
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the substrate concentration to achieve 50% of Vmax, and Ksi is the substrate inhibition
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constant.
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Transport Experiments of Acacetin in the Caco-2 Cell Model. Caco-2 cells
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derived from human colon adenocarcinoma, which structurally and functionally
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resembling the small intestinal epithelium when forming cell monolayer
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laboratory, the Caco-2 cells were routinely cultured in DMEM (10% fetal bovine
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serum, 1% nonessential aminoacids, 1% L-glutamine, and 1% antibiotics (penicillin
183
and streptomycin)), which was replaced with a fresh one every other day. The
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growing atmosphere was 5% CO2 and 90% relative humidity at 37 °C. The cells
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were seeded on 3 μm porous six-well polycarbonate cell culture inserts. The cells
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were ready for experiment at 19-22 days after seeding. Transport experiments on the
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Caco-2 cell model have been reported previously. Briefly, cell monolayers were
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washed three times with HBSS (pH 7.4) at 37 °C. The value of transepithelial
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electrical resistance of cell monolayers was measured and not over 460 Ω/cm2.
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Subsequently, the monolayers were incubated with HBSS for 1 h. Acacetin solution
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(10 μM) was loaded on either apical (AP) or basolateral (BL) side, and the other side
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was loaded with HBSS. Transport experiments were conducted with transporter
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inhibitors (10 μM) including Ko143 (inhibitor of BCRP) and MK571 (inhibitor of
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MRP1 and MRP2). Ko143 was added to AP side, and MK571 was added to AP and
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BL side to study MRP2 and MRP1, respectively. Each sample was prepared in
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triplicate. The samples (500 μL) were collected at different time points (0, 0.5, 1, 1.5,
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2 h), and the same volume of corresponding solution was added each well. 11
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Intracellular concentrations of acacetin sulfate were determined at the end of the
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experiment. Finally, the cell monolayers were removed, pooled with 1 mL HBSS
200
and sonicated for 30 min in an ice bath. All samples were stored in the −20 °C prior
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to measurement by LC–MS/MS. The major parameters including transport amount,
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efflux rate, clearance rate (CL), and fraction of the metabolized dose (Fmet) were
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calculated according to previous study 38.
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Data Analysis. Pharmacokinetic parameters were obtained by noncompartmental oral
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administration mouse model of WinNonlin 3.3. One-way ANOVA with LSD or
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Dunnett's T3 test was applied to evaluate statistical differences. Differences were
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considered significant when p
