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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

ACS Paragon Plus Environment


1

ABSTRACT: Acacetin, an important component of acacia honey, exerts extensive

2

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

8

decreased significantly in multiple drug resistance protein 1 knockout (Mrp1−/−) mice,

9

while increased evidently in breast cancer resistance protein knockout (Bcrp−/−) mice.

10

In Caco-2 monolayers, efflux and clearance of acacetin-7-sulfate reduced distinctly by

11

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

2


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INTRODUCTION

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Honey has been flavorful for many decades because of its high nutritional value

3

and contribution to human health 1. Acacia honey, produced by bees in acacia flowers,

4

is widely consumed all year round in many countries and is the most popular in

5

Malaysia 2. Acacetin, an important flavone in acacia honey, is one of the main

6

flavonoids accounting for 28.83-113.06 mg/kg of whole honey composition

7

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.

15

Glucuronidation for acacetin has been extensively investigated in our laboratory 11, 12

16

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

19

various important functions with distinct tissue distribution for each individual

20

using the universal sulfonyl donor molecule 3'-phosphoadenosine-5'-phosphosulfate

21

(PAPS) to form sulfate

22

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

3



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23

regulating important biological processes including blood clotting, formation of

24

connective tissues, and functionality of secreted proteins, hormones and signaling

25

molecules 16.

26

The activation and inactivation of numerous xenobiotics and endogenous

27

compounds occur by sulfonation pathway, which may change the physiological

28

function of body. For instance, the sulfonation of certain drugs may enhance

29

therapeutic activity. Minoxidil is a drug for antihypertensive and hair growth

30

stimulating, and tamoxifen is a common drug for treating breast cancer, both of their

31

sulfate metabolite are responsible for their biological activities

32

sulfonation is the most abundant post-translational modification of tyrosine residues

33

implicated in numerous physiological and pathological processes

34

acts as a determinant of protein–protein interactions, which are involved in leukocyte

35

adhesion, hemostasis and receptor-mediated signaling

36

essential for proper blood clotting in response to vessel injuries and binding of

37

chemokines to their receptors CCR5 and CXCR4

38

showed that individual differences in various genes of sulfonation pathway may

39

contribute to carcinogenesis and patient survival. For example, polymorphisms in

40

SULT1E1, which involves in estrogen sulfonation, are related to the survival of

41

patients with estrogen-dependent cancers. Hirata et al. and Rebbeck et al. found that

42

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


24

.

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45

Previous reports also showed that many flavonoid conjugates are substrates of efflux

46

transporters (e.g., p-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and

47

multiple drug resistance proteins (MRPs)), which are expressed abundantly in the

48

intestine and liver

49

determined by sulfotransferases, efflux transporters or both and which efflux

50

transporters and sulfotransferases involved in this process remain unknown. Hence,

51

elucidation of sulfonation disposition of acacetin in vivo could help gain a further

52

systematic understanding of acacetin metabolic and disposition characteristics in vivo.

53

To further understand the sulfonation disposition of acacetin, incubation in vitro,

54

pharmacokinetics and Caco-2 cell monolayer models were utilized in the present

55

study. A sensitive and reliable LC-MS/MS method was developed to determine

56

acacetin and its sulfate accurately, directly and simultaneously. The metabolic

57

characteristics of species and isoforms were used to study the sulfonation activity of

58

different species and ascertain major isoform mediating acacetin sulfonation.

59

Pharmacokinetics of knockout mice and wild-type mice provided insights into the

60

effect of efflux transporters on the exposure level of acacetin sulfate. The Caco-2 cell

61

model combined with selective inhibitors was used as an in vitro approach to confirm

62

the role of efflux transporters.

63

MATERIALS AND METHODS

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Chemicals and Reagents. Acacetin (≥98%, HPLC grade) was purchased from

65

Chengdu Must Biotechnology Co., Ltd. (Chengdu, China). Acacetin-7-sulfate

66

(Aca-7-S) was purified from incubation in vitro and identified using electrospray

25

. However, whether the sulfonation disposition of acacetin is

5



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67

ionization mass spectrometry and diode array detector (DAD) as previously

68

described

69

Merck Company (Merck Millipore, USA). Magnesium chloride, dimethyl sulfoxide

70

(DMSO), β-cyclodextrins and PAPS were purchased from Sigma-Aldrich (St. Louis,

71

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

74

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

78

analytical grade and used as received.

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Animals. Wild-type FVB mice were purchased from Vital river Co., Ltd (Beijing,

80

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.

83

LC-MS/MS Method Development. Agilent 6540 Accurate-Mass quadrupole time

84

of flight (Q-TOF) MS System and Agilent 6490 QQQ MS tandem Agilent 1290

85

UHPLC System were used for qualification and quantification of acacetin and its

86

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

88

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

6


27, 28

, which mainly

) of wild-type FVB and

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89

gradient: 0–1.5 min, 30%B, 1.5–2.5 min, 30%–70% B, 2.5–3.5 min, 70%–100% B,

90

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.

94

Sheath gas flow rate and gas flow rate of 11 and 14 L/min, respectively. Injection

95

volume: 5 μL; column temperature: 35 °C. Data acquisition and analysis were

96

performed using Agilent Mass hunter software. The observed compound m/z value

97

were as follows: Aca-7-S (363.1→282.9); testosterone (289.01→97.2); and acacetin

98

(285→242).

99

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

101

environmentally controlled room (temperature: 25 ± 2 °C, humidity: 50 ± 5%, and

102

12 h dark-light cycle) for at least three days prior to the experiments. The S9

103

fractions were prepared using a procedure published previously with minor

104

modification

105

centrifuge tubes at −80 °C. And the S9 fractions protein concentration was

106

determined by the Bio-Rad protein assay (Bio-Rad Laboratories, USA) using bovine

107

serum albumin as the standard.

108

Species-dependent Sulfonation of Acacetin in Liver S9 Fractions of Different

109

Species (Human, Monkey, Dog, Rat and Mouse). The sulfonation reaction system

110

and incubation procedures were the same as those previous study

30-32

. The liver, intestine and colon S9 fractions of mice were stored in

7


33

. Briefly, the


111

reaction mixture (total volume 200 μL) consists of 10 μL enzymes (liver S9 fractions

112

of species, 0.025 mg/mL), 5 μL magnesium chloride (1 mM), 5 μL PAPS (0.05 mM),

113

178 μL KPI buffer (44.5 mM, pH 7.4) and 2μL acacetin (3, 6, and 24 nM). The S9

114

fractions or substrate must be added lastly. The mixture prepared in equal triplicates

115

was incubated in a 37 °C shaking water bath (speed=50 rpm) for 20-30 min. The

116

metabolic percentage of acacetin was not over 30%. Finally, the reactions were

117

stopped by adding a semisystem volume of the testosterone acetonitrile solution. The

118

mixture system was vortexed for 3min, and centrifuged at 19375 × g for 30 min,

119

4 °C. The supernatant was directly subjected to LC–MS/MS for analysis.

120

SULT Isoform-specific Metabolic Characteristics of Acacetin by Recombinant

121

Human SULTs (1A1, 1B1, 1E1, 2A1 and 2B1). The sulfonation reaction system and

122

incubation procedures were identical to the system for species-dependent sulfonation

123

of acacetin. Briefly, the reaction mixture (total volume 200 μL) with acacetin (3, 6,

124

and 24 nM) was mediated by SULT isoforms (0.0025 mg/mL). Time for incubation

125

was 20-30 min. The metabolic percentage of acacetin was also not over 30%. Lastly,

126

the reactions were stopped by the testosterone acetonitrile solution. The mixture

127

system was centrifuged at 19375 × g for 30 min, 4 °C, and the supernatant was

128

analyzed directly by LC–MS/MS.

129

Pharmacokinetic Studies in the Wild-type, Bcrp−/−, Mrp1−/− and Mrp2−/− FVB

130

Mice. The FVB mice were used extensively in transgenic research because of its

131

defined inbred background (high gene homology and little individual difference),

132

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

134

Bcrp−/−, Mrp1−/− and Mrp2−/− mice in our study. The male wild-type, Bcrp−/−, Mrp1−/−

135

and Mrp2−/− FVB mice were kept in an environmentally controlled room for at least

136

1 week prior to the experiments. Animal experiments were performed in accordance

137

with the Guide for the Care and Use of Laboratory Animals by the National

138

Institutes of Health, and the procedures were approved by the Ethical Committee of

139

Guangzhou University of Chinese Medicine (Guangzhou, China). The acacetin

140

solution system consisted of 5% DMSO, 5% ethanol and 90% β-cyclodextrin

141

aqueous solution (1g β-cyclodextrins: 4 mL 0.9% normal saline) at 0.5 mg/mL. Prior

142

to pharmacokinetic experiments, the wild-type, Bcrp−/−, Mrp1−/− and Mrp2−/− FVB

143

mice (n=5) were fast for at least 8 h. The acacetin solution was administered orally

144

to the mice at a dose of 5 mg/kg. Blood samples (about 30 μL) of wild-type, Bcrp−/−,

145

Mrp1−/− and Mrp2−/− FVB mice were collected from 5 mice of the same strain at

146

each time point by the tail vein to heparinized tubes at 0, 3, 5, 10, 15, 20, 30, 60, 180,

147

300, 420, 540, 720 and 1440 min, and then centrifuged at 11481×g for 8 min at 4 °C.

148

Afterward the plasma supernatant were collected and stored at −20 °C until analysis.

149

Enzyme Kinetic Studies of Acacetin Sulfonation by Mouse Liver, Small

150

Intestine and Colon S9 Fractions. The sulfonation reaction system and incubation

151

procedures were also identical to the system of species-dependent acacetin

152

sulfonation. Briefly, the reaction mixture (total volume 200 μL) with acacetin

153

(0.005-20 μM) was mediated by S9 fractions (0.025-0.05 mg/mL) from mouse. Time

154

for incubation was 15-30 min. Formation rates of acacetin sulfonation were 9



Page 10 of 43

155

determined by the amounts of metabolite formed per mg protein per min

156

(pmol/mg/min). The data were obtained from triplicate reactions. Kinetic parameters

157

estimated by fitting the initial rate to the rate equations and by subsequent nonlinear

158

least-squares regression were obtained according to the profile of Eadie-Hofstee

159

plots

160

Aca-7-S at respective substrate concentrations (C) were suitable for the standard

161

Michaelis-Menten equation:

34, 35

. When the Eadie-Hofstee plot was linear, the formation rates (V) of

𝑉=

𝑉𝑚𝑎𝑥 × 𝐶 𝐾𝑚 + 𝐶

162

Where Km is the Michaelis-Menten equation constant, and Vmax is the maximum rate

163

of sulfate formation. When Eadie-Hofstee plots displayed characteristic profiles of

164

atypical kinetics 34, 36, data from these atypical profiles were suitable for Eq (1) and (2)

165

by using the ADAPT II program. To confirm the best-fit model, model candidates

166

were assessed using the Akaike's information criterion (AIC) and R2 value

167

Therefore, using this minimum AIC estimation, a negative AIC value is considered a

168

better indication of data compared with that of a data set with a positive AIC value.

169

Reaction rate =

[𝑉𝑚𝑎𝑥 −0 +𝑉𝑚𝑎𝑥 −𝑑 (1−𝑒 −𝐶𝑅 )]×𝐶 𝐾𝑚 +𝐶

34

.

(1)

170

Where Vmax-0 is the maximal intrinsic enzyme activity, Vmax-d is the maximal inducible

171

enzyme activity, R is the rate of enzyme activity induction, C is the concentration of

172

substrate, and Km is the substrate concentration to achieve 50% of (Vmax-0 + Vmax-d).

173

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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176

the substrate concentration to achieve 50% of Vmax, and Ksi is the substrate inhibition

177

constant.

178

Transport Experiments of Acacetin in the Caco-2 Cell Model. Caco-2 cells

179

derived from human colon adenocarcinoma, which structurally and functionally

180

resembling the small intestinal epithelium when forming cell monolayer

181

laboratory, the Caco-2 cells were routinely cultured in DMEM (10% fetal bovine

182

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

184

growing atmosphere was 5% CO2 and 90% relative humidity at 37 °C. The cells

185

were seeded on 3 μm porous six-well polycarbonate cell culture inserts. The cells

186

were ready for experiment at 19-22 days after seeding. Transport experiments on the

187

Caco-2 cell model have been reported previously. Briefly, cell monolayers were

188

washed three times with HBSS (pH 7.4) at 37 °C. The value of transepithelial

189

electrical resistance of cell monolayers was measured and not over 460 Ω/cm2.

190

Subsequently, the monolayers were incubated with HBSS for 1 h. Acacetin solution

191

(10 μM) was loaded on either apical (AP) or basolateral (BL) side, and the other side

192

was loaded with HBSS. Transport experiments were conducted with transporter

193

inhibitors (10 μM) including Ko143 (inhibitor of BCRP) and MK571 (inhibitor of

194

MRP1 and MRP2). Ko143 was added to AP side, and MK571 was added to AP and

195

BL side to study MRP2 and MRP1, respectively. Each sample was prepared in

196

triplicate. The samples (500 μL) were collected at different time points (0, 0.5, 1, 1.5,

197

2 h), and the same volume of corresponding solution was added each well. 11


37

. In our


198

Intracellular concentrations of acacetin sulfate were determined at the end of the

199

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

201

to measurement by LC–MS/MS. The major parameters including transport amount,

202

efflux rate, clearance rate (CL), and fraction of the metabolized dose (Fmet) were

203

calculated according to previous study 38.

204

Data Analysis. Pharmacokinetic parameters were obtained by noncompartmental oral

205

administration mouse model of WinNonlin 3.3. One-way ANOVA with LSD or

206

Dunnett's T3 test was applied to evaluate statistical differences. Differences were

207

considered significant when p

Sulfonation Disposition of Acacetin: In Vitro and in Vivo - American

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