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Cysteine-based Protein Adduction by EpoxideDerived Metabolite(s) of Benzbromarone Hui Wang, Yukun Feng, Qian Wang, Xiucai Guo, Wenlin Huang, Ying Peng, and Jiang Zheng Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00275 • Publication Date (Web): 16 Nov 2016 Downloaded from http://pubs.acs.org on November 21, 2016

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Chemical Research in Toxicology

Cysteine-based Protein Adduction by Epoxide-Derived Metabolite(s) of Benzbromarone †

†

†

†

θ

†

Hui Wang, Yukun Feng, Qian Wang, Xiucai Guo, Wenlin Huang , Ying Peng, * and Jiang ‡¶

Zheng * †

‡

College of Pharmacy, Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang, Liaoning, 110016, P. R. China θ

Department of Biochemistry, University of Washington, Seattle, WA 98195， USA

¶

Key Laboratory of Pharmaceutics of Guizhou Province, Guizhou Medical University, Guiyang, Guizhou, 550004, P. R. China

Running title: Benzbromarone-derived protein adduction

Corresponding Authors: Jiang Zheng, PhD Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang, Liaoning, 110016, P. R. China Key Laboratory of Pharmaceutics of Guizhou Province, Guizhou Medical University, Guiyang, Guizhou, 550004, P. R. China Email: [email protected] Tel: 206-884-7651; Fax: 206-987-7660

Ying Peng, PhD School of Pharmacy, Shenyang Pharmaceutical University, PO Box 21, 103 Wenhua Rode, Shenyang, 110016, P. R. China Email: [email protected] Tel: +86-24-23986361; Fax: +86-24-23986510

ACS Paragon Plus Environment


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TOC Graphic

P450 3A

Cys

H2O

Protein modification


Hepatotoxicity

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ABSTRACT Benzbromarone (BBR) is a therapeutically useful uricosuric agent but can also cause acute liver injury.

The hepatotoxicity of BBR is suggested to be associated

with its metabolic activation.

Our recent metabolic study demonstrated that BBR

was metabolized to epoxide intermediate(s) by cytochromes P450 3A and the intermediate(s) was reactive to N-acetylcysteine.

The objectives of the present study

were to determine the chemical identity of the interaction of protein with the epoxide intermediate(s) of BBR and to define the association of the protein modification with hepatotoxicity induced by BBR.

Microsomal incubation study showed that the

reactive intermediate(s) covalently modified microsomal protein at cysteine residues. Such adduction was also observed in hepatic protein obtained from liver of mice given BBR. manners.

The protein covalent binding occurred in time- and dose-dependent Pretreatment with ketoconazole attenuated BBR-induced protein

modification and hepatotoxicity, while pretreatment with dexamethasone or buthionine sulfoximine potentiated the protein adduction and hepatotoxicity induced by BBR.

A good correlation was observed between BBR-induced hepatotoxicity

and the epoxide-derived hepatic protein modification in mice.

The present study

provided in-depth mechanistic insight into BBR-induced hepatotoxicity.



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INTRODUCTION Gout is a medical condition, known as hyperuricemia, commonly associated with repeated episodes of acute inflammatory arthritis caused by elevated urate blood levels, and urate crystallizes and deposits into joints and/or surrounding tissues.1-3 Benzbromarone (BBR) is a benzofuran derivative clinically used as a uricosuric for the prophylaxis of acute gout attacks.

BBR effectively reduces serum urate levels by

inhibiting a urate reabsorption transporter known as renal urate transporter 1 (URAT1).4,5

In addition, 6-hydroxy BBR, a metabolite of BBR, has also been

reported to show potent hURAT1 inhibition property.6 For many years, BBR was considered to be both effective and well tolerated, with most people achieving normal uric acid levels.

However, after several reports of idiosyncratic liver injury

including some fatalities related to BBR,7-9 the drug had to be withdrawn from the market in Europe. The mechanisms underlying idiosyncratic hepatotoxicity remain largely unknown. It is widely accepted that drug-induced idiosyncratic hepatotoxicity is often associated with metabolic activation mediated by cytochromes P450.10-13 Our recent study demonstrated that BBR was metabolized to epoxide intermediate(s) (Scheme 1).14 Decreased urinary mercapturic acid derived from the epoxide intermediate(s) was observed in ketoconazole pretreated mice after exposure to BBR,14 indicating that P450 3A responded the metabolic activation of BBR.

McDonald and Rettie

proposed the bioactivation of BBR through sequential steps of oxidation of the benzofuran ring to an ortho-quinone intermediate.15 Both of the quinone and epoxide


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intermediates may contribute to the hepatotoxicity exhibited by BBR. Numerous studies have shown that epoxide-derived metabolites of many pro-toxicants can react with nucleophilic sites in protein to form stable protein covalent adduction, possibly triggering various toxicities.

Epoxide metabolite of

naphthalene was responsible for covalent binding to sulfur nucleophiles of protein and was associated with naphthalene-induced Clara cell toxicity.16 Association between styrene- and bromobenzene-induced liver injury and protein adduction through their epoxide intermediate(s) was also established.17-20

All these results suggested that

protein covalent modification by reactive metabolites might contribute to the observed toxicities of the xenobiotics.

Recently, we detected urinary a mercatpturic acid

derived from epoxide metabolites of BBR.14 This led us to reason that the epoxide intermediate(s) of BBR may react with the free thiol groups of proteins to form protein covalent binding and that the protein modification possibly triggers the development of toxicity. To this end, little effort has been directed toward the understanding of the molecular details involved in BBR-derived protein covalent binding.

The objectives

of this study were to define the chemical identity of the interaction of protein with the epoxide-derived metabolite(s) of BBR, to determine the role of P450 3A in BBR-induced protein modification, and to investigate the correlation between the protein adduction and liver injury induced by BBR.



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EXPERIMENTAL PROCEDURES Chemicals and Materials.

Benzbromarone (BBR, >98%) was obtained from

Aladdin Industrial Technology Co., Ltd. (Shanghai, China).

Pronase E ( >98%) was

acquired from Shanghai Yuanye Biological Technology Co., Ltd. (Shanghai, China). Monobromobimane (mBrB, >97%), ketoconazole (KTC, >99%), buthionine sulfoximine (BSO, >99%), DL-dithiothreitol (DTT), L-cysteine, chymotrypsin, S-hexylglutathione, and reduced nicotinamide adenine dinucleotide phosphate (NADPH) were purchased from Sigma-Aldrich (St. Louis, MO).

Dexamethasone

(DEX, >99%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Shenyang, China).

Propranolol was

purchased from Jiangsu Campbell Pharmaceutical Co., Ltd. (Jiangsu, China). organic solvents were from Fisher Scientific

(Springfield, NJ).

All

All reagents and

solvents were of either analytical or HPLC grade.

Protein Modification and Digestion. A stock solution of BBR was prepared in acetonitrile.

The incubation mixtures were performed in a final volume of 0.5 mL

phosphate buffer (pH 7.4) containing mouse liver microsomes (1.0 mg protein/mL, 125 µL ), BBR (100 µM), and MgCl2 (3.2 mM). addition of NADPH (1.0 mM).

The reactions were initiated by

After 1 h incubation at 37 °C, the resulting protein

samples were denatured by heating at 60 °C for 30 min, followed by centrifugation at 16,000 g for 10 min. The resulting pellets were suspended in 50 mM ammonium bicarbonate (pH 8.0) and mixed with 5.0 mM DTT.

After 1 h incubation at 60 °C,

the protein samples were digested with a mixture of chymotrypsin (1.0 mg/mL) and


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Pronase E (2.0 mg/mL) in the presence of CaCl2 (5.0 mM) with continuous incubation at 37 °C for 15 h.21,22 Each incubation was performed in duplicate.

The digestion

mixtures were centrifuged at 16,000 g for 10 min, and the supernatants were mixed with 10 µL of S-hexylglutathione dissolved in acetonitrile (6.39 nM, internal standard, IS) before subjecting onto LC-MS/MS system for analysis.

Chemical Synthesis of 6-Cysteine BBR Conjugate (A1). Cysteine adduct A1 was synthesized by enzymatic hydrolysis of 6-NAC BBR (Scheme 2) which is an N-acetylcysteine (NAC) conjugate derived from epoxide intermediate(s) of BBR, and the NAC conjugate had been synthesized in our laboratory.14

6-NAC BBR (0.5 mg)

dissolved in 50 µL of acetonitrile was added to a solution (1.0 mL) containing Pronase E (2.0 mg/mL) and CaCl2 (5.0 mM).

The mixture was incubated at 37 °C

for 4 h, and then the digestion mixtures were centrifuged at 16,000 g for 10 min, and the supernatants were subjected onto an LC-MS/MS system for analysis.

Animals and Treatment. Male Kunming mice (20 ± 2 g) were purchased from the Animal Center of Shenyang Pharmaceutical University (Shenyang, China).

Animal

maintenance and treatment met the protocols approved by the Ethics Review Committee for Animal Experimentation of Shenyang Pharmaceutical University. Mice had free access to food and water and were kept in a controlled environment (temperature of 25 °C and moderate humidity) under 12 hours dark/light cycle for at least 5 days for quarantine. After fasting for 12 h with free access to water prior to the experiment, mice



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were treated intraperitoneally (i.p.) with BBR dissolved in corn oil at doses of 0, 30, 50, or 100 mg/kg.

Liver tissues were harvested 30 min after the treatment

(dose-dependent experiment) or after 10 min, 30 min, 1 h, 2 h, 4 h, and 8 h (time-dependent experiment conducted at the dosage of 50 mg/kg).

Before

collecting the liver tissues, the animals were anesthetized with diethyl ether. In a separate study, mice were randomly divided into three groups (n = 4).

One

group of mice were pretreated (i.p.) with dexamethasone (DEX, 50 mg/kg) for 5 consecutive days, then the animals were given (i.p.) BBR at 50 mg/kg or vehicle. The other two groups of mice were treated (i.p.) with ketoconazole (KTC, 100 mg/kg) in corn oil or buthionine sulfoximine (BSO, 666 mg/kg) in saline, respectively. After 1.5 h, the animals were given BBR at dose of 50 mg/kg or vehicle. of BBR alone excluded DEX, KTC or BSO pretreatment. anesthetized with diethyl ether.

The group

Animals were

The blood samples were harvested by cardiac

puncture and liver tissues were collected at 1 h following BBR treatment.

Sample Preparation and AST Assay.

The collected blood samples were

allowed to clot at room temperature for 3 h, followed by centrifuging at 8,000 g for 10 min.

The resulting sera were collected for AST assay on a VITROS® 5600

Integrated System (Ortho-Clinical Diagnostics, Rochester, NY), and the serum AST activity was used to evaluate liver injury.

The liver tissues (0.2 g) were

homogenized in 2.0 mL phosphate buffer (0.1 M, pH 7.4).

The resulting tissue

homogenates were denatured at 60 °C for 30 min, and then digested with a mixture of chymotrypsin and Pronase E as described above.

To ensure that A1 resulted from


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hepatic proteins, the above liver homogenates were denatured by heating in a water bath at 60 °C for 30 min, followed by overnight incubation with chymotrypsin (1.0 mg/mL) at 37 °C for proteolytic digestion.

Toxicokinetic Study. Mice were randomly divided into two groups, each group included 36 mice, and each time point contained 4 mice.

Mice in group I were given

BBR (50 mg/kg, i.p.) alone, and mice in group II were pretreated with KTC and then received BBR (50 mg/kg, i.p.) 1.5 h after the pretreatment.

Blood samples were

collected by cardiac puncture at time intervals of 10, 20, 30 min, and 1, 2, 4, 6, 8, and 12 h after BBR injection.

The control animals treated with corn oil were included.

Aliquots (30 µL) of plasma prepared as described above were mixed with 90 µL of ice-cold acetonitrile containing propranolol (3.22 µM) as the IS.

The resulting

mixtures were vortex-mixed and centrifuged at 16,000 g for 10 min to remove the precipitated protein.

The resultant supernatants (2 µL) were injected into

LC-MS/MS system for analysis.

A non-compartmental model was utilized to

describe the plasma concentration-time profiles of BBR.

The values of maximum

concentration (Cmax), time required to reach maximum concentration (Tmax), area under the concentration-time profiles (AUC), half life (t1/2), apparent volume of distribution (Vz/F), and clearance rate (CLz/F) were determined.

Assessment of Hepatic and Plasma GSH Contents. Mice with the same treatments as the toxicokinetic study were used for the assessment of hepatic and plasma GSH contents.

Plasma and liver samples were harvested at time intervals of 10, 20, 30, 45



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min, and 1, 2, 6, and 12 h after BBR treatment.

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The biological samples were

prepared as described above for GSH content assessment, according to our published method.23

Briefly, 60 µL of GSH solutions in phosphate-buffered saline (pH 7.4) or

GSH-containing biological samples were added to Eppendorf vials containing 20 µL of monobromobimane (mBrB, 1.38 mM in acetonitrile) solution.

The derivatization

reaction was carried out at room temperature in the dark for 20 min to produce mBrB-GSH conjugate, followed by addition of 100 µL 10% 5-sulfosalicylic acid to precipitate protein.

The resulting samples were spiked with 20 µL of

S-hexylglutathione dissolved in acetonitrile (1.28 nM, IS).

The mixtures were

centrifuged at 16,000 g for 10 min, and 2.0 µL of the resulting supernatants were injected into LC-MS/MS system for analysis.

LC-MS/MS Method.

LC-MS/MS analyses were performed on an AB SCIEX

Instruments 5500 triple quadrupole (Applied Biosystems, Foster City, CA) interfaced online with an Agilent 1260 Series Rapid Resolution LC system (Agilent Technologies, Santa Clara, CA).

Samples were analyzed by multiple-reaction

monitoring (MRM) scanning in positive ion mode.

The conditions of the Turbo Ion

Spray interface for electrospray ionization: ion spray voltage, 5,500 V; source temperature, 650 °C; curtain gas, 35 psi; ion source gas 1, 50 psi; ion source gas 2, 50 psi.

Data were analyzed by Applied Biosystems/SCIEX Analyst software (version

1.6.2). For the analysis of A1, the chromatographic separation was achieved on an Ultimate XB-C18 column (2.1 × 100 mm, 3 µm, Welch Scientific, Inc., Shanghai,


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China), using 0.1% (v/v) formic acid in acetonitrile and 0.1% (v/v) formic acid in water as mobile phases A and B, respectively. The gradient elution started at 10% A for 2 min, ramped to 100% A over 12 min, held at 100% A for 2 min, and then returned to 10% A in 1 min to equilibrate the column. and the column temperature was 25 °C.

The flow rate was 0.4 mL/min,

The parameters of ion pairs (declustering

potential, DP; collision energy, CE; collision cell exit potential, CXP) were m/z 544→279 (110, 25, 10) for A1, and m/z 392.2→246.3 (86, 24, 5) for S-hexylglutathione (IS for A1). The proteolytic digestion products were analyzed by LC-MS/MS in full scan mode.

The chromatographic separation was performed on a VYDAC C4 column

(5.0 µm, 250 mm× 4.6 mm; San Jose, CA), using 0.1% (v/v) formic acid in water and 0.1% (v/v) formic acid in acetonitrile as mobile phases A and B.

The gradient

elution started at 5% B for 10 min, ramped to 30% B over 40 min and to 95% B in 10 min, and then returned to 5% B in 2 min to equilibrate the column.

The flow rate

was 0.8 mL/min, and the column temperature was set at 25 °C. For the analysis of mBrB-GSH conjugate and plasma BBR concentrations, the chromatographic separation was performed on a BDS HYPERSIL C18 ODS column (5.0 µm, 150 mm× 4.6 mm; Thermo, San Jose, CA, USA), using 0.1% (v/v) formic acid in water and 0.1% (v/v) formic acid in acetonitrile as mobile phases A and B, respectively.

The same gradient elution was applied for analytical separation of

mBrB-GSH conjugate and BBR: 0-1 min, 10% B; 1-6 min, 10-90% B; 6-9 min, 90-10% B; 9-10 min, 10% B.

The flow rate was 1.0 mL/min, and the column



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temperature was 25 °C.

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The parameters of ion pairs (DP, CE, CXP) were m/z

498.2→192.2 (115, 50, 3) for mBrB-GSH, m/z 392.2→246.3 (86, 24, 5) for S-hexylglutathione (IS for mBrB-GSH ), m/z 425→279 (110, 35, 3) for BBR, and m/z 260.7→116.3 (75, 25, 10) for propranolol (IS for BBR) as employed in our early study.14 It should be noted that MS/MS spectra were obtained from an AB SCIEX Instruments 4000 Q-Trap (Applied Biosystems, Foster City, CA) interfaced online with an Agilent 1260 Series Rapid Resolution LC system (Agilent Technologies, Santa Clara, CA).

The information-dependent acquisition (IDA) method was

utilized to trigger the enhanced product ion (EPI) scans by analyzing MRM signals. The EPI scan was run in positive mode at a scan range for productions from m/z 100 to 600.

The collision energy was set at 40 eV with a spread of 15 eV.

The other

parameters of LC-MS/MS were in agreement with the conditions described above except for the curtain gas (25 psi).

Statistic Analysis. Drug and Statistic (DAS) 2.1.1 software (Mathematical Pharmacology Professional Committee of China, Shanghai, China) was used for profiling the toxicokinetic data.

Statistical analyses were performed by unpaired Student's t-tests,

using Graph Pad Prism software.

A p value of less than 0.05, 0.01 or 0.001 was

considered significant.


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RESULTS Cysteine-based Protein Adduction by Epoxide Metabolite(s) of BBR In Vitro. BBR was incubated with mouse liver microsomes, and the resulting microsomal proteins were exhaustively digested by proteinase.

A product with the retention time at 10.0

min was detected with a molecular cluster of m/z 542 (50%), 544 (100%), and 546 (50%) (Figure 1A).

The observed value of the molecular ions and the pattern of the

triplets suggest that the product was an adduct, namely A1, composed of a molecule of cysteine and a molecule of BBR.

No such adduct was detected in the digestion

mixtures from the microsomal incubation system in the absence of NADPH (data not shown).

The MS/MS spectrum of A1 (Figure 1D) obtained by MRM-EPI scanning

displayed the indicative characteristic fragment ions of a BBR-cysteine adduct, such as product ion m/z 455 resulting from the cleavage of the carbon-sulfur bond within the cysteine moiety (89 Da) and product ion m/z 423 arising from the loss of the whole cysteine (121 Da), respectively.

The fragment ion of A1 at m/z 279 (loss of

C7H3Br2O2) observed was also found in the mass spectrum of parent BBR we recently reported,14 indicating that the dibromohydroxybenzoyl ring retained intact.

The

product ion at m/z 264 corresponded to a loss of dibromohydroxybenzoyl ring of A1, leading us to propose that the cysteine was attached to the benzofuran ring.

In

addition, our recent study demonstrated that BBR was metabolized to epoxide intermediate(s) (Scheme 1) which can be captured by NAC to form 6-NAC BBR. The observed A1 shared similar mass spectrometric identities with 6-NAC BBR. Thus, it is most likely that the protein adduction was derived from the epoxide



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intermediate(s) of BBR (Scheme 1).

Identification of 6-Cysteine BBR Conjugate (A1).

Adduct A1 was further

characterized by enzymatic hydrolysis of 6-NAC BBR (Scheme 2) previously prepared in our laboratory.14 One product was observed with the same retention time (Figure 1C) and mass spectrometric identities (Figure 1F) of A1.

The 2-D NMR

spectrum of the NAC conjugate demonstrated that the NAC was attached to position C6 of BBR.14

This led us to conclude that A1 was a BBR-derived cysteine conjugate

whose C6 is attached with a molecule of cysteine, indicating that the sulfhydryl group of cysteine residues of protein attacked the epoxide moiety at C6 of the epoxide metabolite(s) of BBR (Scheme 1).

Cysteine-based Protein Adduction by Epoxide Metabolite(s) of BBR In Vivo. The success in the in vitro study above encouraged us to expand the protein adduction detection to animal work.

Mice were administered with BBR at 50 mg/kg, and liver

tissues were harvested 30 min after the treatment, followed by proteolytic digestion with a mixture of chymotripsin and Pronase E, and LC-MS/MS analysis.

One

product with the same chromatographic and mass spectrometric properties of A1 (Figure 1B) was detected in the resulting digestion mixtures.

No such product was

observed in the digested samples obtained from the liver of animals treated with vehicle (data not shown).

Further more, we examined the time course and dose

dependence of the hepatic protein adduction in mice treated with BBR.

The most

abundant cysteine-based adduction detected in mouse liver was normalized to 100%.


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The hepatic protein adduction arising from cysteine-modification reached its peak at 0.5 h after the administration (Figure 2).

In addition, the protein adduction was

found to elevate with the increase in the doses of BBR given in mice (Figure 3). In a separate study, proteins precipitated from the liver homogenates obtained from BBR-treated mice were digested with chymotrypsin.

We found several

peptides (protein digestion products) demonstrated the characteristic mass spectrometric pattern of the bromines as shown in Figure S1 and S2. signals were detected in those of vehicle-treated animals.

No such

The results conformed

protein adduction resulting from the metabolic activation of BBR.

Correlation between Hepatic Protein Adduction and Liver Injury Induced by BBR. Our recent study demonstrated that BBR was bioactivated by P450 3A to the epoxide intermediate(s).14

To investigate the involvement of P450 3A-mediated

metabolism in BBR-induced hepatotoxicity, we examined the effect of KTC (a P450 3A inhibitor) and DEX (a P450 3A inducer) on BBR-induced liver toxicity by monitoring serum AST activity.

A single dose of BBR at 50 mg/kg caused elevation

(p < 0.05) of serum AST activity (134 U/L; 102 U/L for control) in mice 1 h following administration.

Pretreatment with KTC mildly attenuated the elevation of serum

AST activity (from 134 to 120 U/L) induced by BBR.

A significant increase (p

Cysteine-Based Protein Adduction by Epoxide ... - ACS Publications

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