Glutathione conjugation and protein adduction derived from oxidative debromination of benzbromarone in mice

Article abstract of DOI:10.1124/dmd.119.087460

Benzbromarone (BBR), a uricosuric agent, has been known to induce hepatotoxicity, and its toxicity has a close relation to cytochrome P450-mediated metabolic activation. An oxidative debromination metabolite of BBR has been reported in microsomal incubations. The present study attempted to define the oxidative debromination pathway of BBR in vivo. One urinary mercapturic acid (M1) and one glutathione (GSH) conjugate (M2) derived from the oxidative debromination metabolitewere detected in BBR-treated mice after solid phase extraction.M1 andM2 shared the same chromatographic behavior and mass spectral identities as those detected in N-acetylcysteine/GSHand BBR-fortified microsomal incubations. The structure of M1 was characterized by chemical synthesis, along with mass spectrometry analysis. In addition, hepatic protein modification that occurs at cysteine residues (M93) was observed in mice given BBR. The observed protein adduction reached its peak 4 hours after administration and occurred in a dose-dependent manner. A GSH conjugate derived from oxidative debromination of BBR was detected in livers of mice treated with BBR, and the formation of the GSH conjugate apparently took place earlier than the protein adduction. In summary, our in vivo work provided strong evidence for the proposed oxidative debromination pathway of BBR, which facilitates the understanding of the mechanismsof BBR-induced hepatotoxicity. SIGNIFICANCE STATEMENT This study investigated the oxidative debromination pathway of benzbromarone (BBR) in vivo. One urinary mercapturic acid (M1) and one glutathione (GSH) conjugate (M2) derived from the oxidative debromination metabolite were detected in BBR-treated mice. M1 and M2 were also observed in microsomal incubations. The structure of M1 was characterized by chemical synthesis followed by mass spectrometry analyses. More importantly, protein adduction derived fromoxidative debromination ofBBR(M93) was observed in mice given BBR, and occurred in dose- and time-dependent manners. The success in detection of GSH conjugate, urinary N-acetylcysteine conjugate, and hepatic protein adduction in mice given BBR provided solid evidence for in vivo oxidative debromination of BBR. The studies allowed a better understanding of the metabolic activation of BBR.

Full text of DOI:10.1124/dmd.119.087460

Supplemental material to this article can be found at:
http://dmd.aspetjournals.org/content/suppl/2019/09/04/dmd.119.087460.DC1
1521-009X/47/11/1281–1290$35.00
DRUG METABOLISM AND DISPOSITION
https://doi.org/10.1124/dmd.119.087460
Drug Metab Dispos 47:1281–1290, November 2019
Copyright ª 2019 by The American Society for Pharmacology and Experimental Therapeutics
Glutathione Conjugation and Protein Adduction Derived from
Oxidative Debromination of Benzbromarone in Mice s
Hui Wang, Wenbao Wang, Bowen Gong, Zedan Wang, Yukun Feng, Weige Zhang, Shaojie Wang,
Ying Peng, and Jiang Zheng
State Key Laboratory of Functions and Applications of Medicinal Plants, Key Laboratory of Pharmaceutics of Guizhou Province,
Guizhou Medical University, Guiyang, Guizhou, People’s Republic of China (J.Z.); and Wuya College of Innovation (H.W., Y.F., Y.P.,
J.Z.) and Key Laboratory of Structure-Based Drug Design and Discovery (Ministry of Education), School of Pharmaceutical
Engineering (W.W., B.G., Z.W., W.Z., S.W.), Shenyang Pharmaceutical University, Shenyang, Liaoning, People’s Republic of China.
Received April 8, 2019; accepted August 10, 2019
ABSTRACT
Benzbromarone (BBR), a uricosuric agent, has been known to induce In summary, our in vivo work provided strong evidence for the
hepatotoxicity, and its toxicity has a close relation to cytochrome proposed oxidative debromination pathway of BBR, which facilitates
P450–mediated metabolic activation. An oxidative debromination the understanding of the mechanisms of BBR-induced hepatotoxicity.
metabolite of BBR has been reported in microsomal incubations.
SIGNIFICANCE STATEMENT
The present study attempted to define the oxidative debromination
pathway of BBR in vivo. One urinary mercapturic acid (M1) and one This study investigated the oxidative debromination pathway of
glutathione (GSH) conjugate (M2) derived from the oxidative debromi-
nation metabolite were detected in BBR-treated mice after solid phase
extraction. M1 and M2 shared the same chromatographic behavior and
benzbromarone (BBR) in vivo. One urinary mercapturic acid (M1)
and one glutathione (GSH) conjugate (M2) derived from the oxidative
debromination metabolite were detected in BBR-treated mice. M1
mass spectral identities as those detected in N-acetylcysteine/GSH- and M2 were also observed in microsomal incubations. The struc-
and BBR-fortified microsomal incubations. The structure of M1 was
characterized by chemical synthesis, along with mass spectrometry
analysis. In addition, hepatic protein modification that occurs at
cysteine residues (M93) was observed in mice given BBR. The
ture of M1 was characterized by chemical synthesis followed by
mass spectrometry analyses. More importantly, protein adduc-
tion derived from oxidative debromination of BBR (M93) was observed in
mice given BBR, and occurred in dose- and time-dependent manners.
observed protein adduction reached its peak 4 hours after admin- The success in detection of GSH conjugate, urinary N-acetylcysteine
istration and occurred in a dose-dependent manner. A GSH conju- conjugate, and hepatic protein adduction in mice given BBR
gate derived from oxidative debromination of BBR was detected in
provided solid evidence for in vivo oxidative debromination of
livers of mice treated with BBR, and the formation of the GSH BBR. The studies allowed a better understanding of the metabolic
conjugate apparently took place earlier than the protein adduction.
activation of BBR.
Introduction
Tolosa et al., 2018). The mechanisms underlying DILI remain unclear.
It is generally accepted that cytochrome P450–mediated metabolic
activation plays a key role in DILI (Guengerich, 2011; McEneny-King
et al., 2017; Quintanilha et al., 2017). Currently, there are four possible
metabolic pathways available for BBR. McDonald and Rettie (2007)
first suggested that BBR was sequentially oxidized by CYP2C9 to
a catechol which can be further oxidized to a reactive o-quinone
intermediate identified as glutathione (GSH) conjugates in liver micro-
somal systems. Previous work from our laboratory revealed that
BBR was metabolized to an epoxide intermediate(s) through the
initial epoxidation of the benzofuran ring by CYP3A (Wang et al., 2016b).
In our following studies, mercapturic acid excreted from urine and
hepatic protein adduction derived from the epoxide intermediate(s)
were observed in BBR-treated mice, which confirmed the epoxi-
dation metabolism of BBR in vivo (Wang et al., 2016a,b). The
correlation between metabolic epoxidation and hepatotoxicity of
BBR was further determined by manipulating the structure of BBR
through introduction of halogen atoms to the site of CYP3A metabolism
Benzbromarone (BBR) is a potent uricosuric therapeutically used
as an antigout agent (Manger, 2015; Stamp et al., 2016). It is reported
that both BBR and its major hydroxylation metabolite, 6-hydroxyBBR,
are strong inhibitors to human uric acid transporter 1, which is responsible
for urate reabsorption (Iwanaga et al., 2005; Nindita et al., 2010).
However, BBR is not widely available due to concerns about serious
idiosyncratic hepatotoxicity (Hautekeete et al., 1995; Suzuki et al.,
2001; Arai et al., 2002; Haring et al., 2013).
Drug-induced liver injury (DILI) is one of the most important
reasons for drug withdrawal from the market (Chen et al., 2014;
This work was supported in part by the National Natural Science Foundation
of China [Grants 81373471, 81430086, and 81773813], the Natural Science
Foundation of Liaoning Province [Grant 2015020738], and the Scientific Research
Program of Hainan Province [Grant ZDYF2016143].
https://doi.org/10.1124/dmd.119.087460.
s This article has supplemental material available at dmd.aspetjournals.org.
ABBREVIATIONS: Ar-H aromatic-H BBR, benzbromarone; DILI, drug-induced liver injury d doublet; ESI, electrospray ionization; GS, source gas;
GSH, glutathione; IS, internal standard; LC-MS/MS, liquid chromatography coupled to tandem mass spectrometry; MRM, multiple reaction
monitoring; MS, mass spectrometry m multiplet; NAC, N-acetylcysteine s single.
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Wang et al.
Sample Preparation for Liquid Chromatography Coupled to Tandem Mass
Spectrometry Analysis
(Wang et al., 2017). Early metabolic studies described that 6- and
19-hydroxy BBR are two major hydroxylation metabolites of BBR
catalyzed by CYP2C9 and CYP3A4, respectively (Kobayashi et al.,
2012). Recently, Cho et al. (2017) identified novel GSH conjugates
derived from 19,6-dihydroxy BBR in human liver microsomal incuba-
tions. They further found that the formation of hepatic GSH conjugates
was proportional to the elevation of plasma alanine aminotransferase
activity in mice treated with BBR (Yoshida et al., 2017).
For identification of urinary NAC and biliary GSH conjugates in BBR-treated
mice, the collected urine samples (24 hours) or bile samples (1, 2, and 4 hours) as
indicated earlier were pooled. A total of 0.6 ml of urine and 0.15 ml of bile were
then subjected to protein precipitation with triple volumes of cold acetonitrile.
After centrifuging at 19,000g for 10 minutes, the resulting supernatants were
concentrated to dryness in a stream of nitrogen at 25ꢀC. The residues were
redissolved in 200 ml of 0.2% (v/v) acetic acid in water. The pretreated urine or
bile samples were loaded onto a ProElut C18 cartridge (500 mg, 50 mm, 60 Å;
Dikma, Lake Forest, CA) that had been conditioned with 4.0 ml of methanol and
4.0 ml of 0.2% (v/v) acetic acid in water. After a complete passage of the samples,
each cartridge was rinsed with 5.0 ml of 0.2% (v/v) acetic acid in water, then
5.0 ml of 0.2% acetic acid in methanol/water (10/90, v/v). Elution was performed
by adding 10.0 ml of 0.2% acetic acid in methanol/water (90/10, v/v). The pooled
eluates were concentrated to dryness, and the resulting residues were redissolved
in 100 ml of 50% acetonitrile in water. A 10-ml aliquot of the supernatants
was analyzed by liquid chromatography coupled to tandem mass spectrometry
(LC-MS/MS).
For determination of BBR-derived GSH conjugates in mouse liver, liver
samples (0.3 g) were homogenized in 3.0 ml of phosphate buffer (pH 7.4)
followed by centrifuging at 8000g for 10 minutes. A 2.0-ml aliquot of acetonitrile
containing internal standard (IS; 50 ng/ml of propranolol) was added to 1.0 ml of
the supernatants. The mixtures were vortex mixed and then centrifuged at 19,000g
for 10 minutes to precipitate protein. The resulting supernatants were dried
by N2 flow and reconstituted in 100 ml of 50% acetonitrile in water for LC-MS/MS
analysis.
The aforementioned metabolic studies of BBR focused on its benzofuran
ring. In 2015, Kitagawara and coworkers discovered that BBR which
contains a phenolic hydroxyl group could be metabolized to a mono-
debrominated catechol through ipso-substitution of BBR (Kitagawara
et al., 2015). Catechols are susceptible to being oxidized to ortho-quinones,
which are known to be capable of adducting biologic macromolecules
and finally result in toxicities. GSH conjugation is generally considered
as an important pathway to protect against the attack of electrophilic
agents. BBR may also be detoxified by reaction with GSH to form
GSH conjugates in vivo that are mainly excreted in bile. GSH conjugates
are often converted to mercapturic acids through enzymatic hydrolytic
cleavage of glutamic acid and glycine followed by N-acetylation of the
remaining cysteine conjugates before excretion in urine (Poon et al., 2001;
Cosnier et al., 2013). When GSH was depleted, hepatic protein was
prone to being exposed to the electrophiles to form protein adduction.
These mercapturates and protein adduction derived from the oxidative
debromination metabolite of BBR can provide additional insight into its
metabolic pathways. This study aimed to investigate and identify the
ipso-substitution metabolite of BBR in mice, including the correspond-
ing GSH conjugate in bile, mercapturic acid in urine, as well as protein
adduction in liver.
For assessment of BBR-derived protein adductions in mouse liver, experi-
ments were carried out according to our established method (Wang et al.,
2016a). In brief, liver homogenates prepared as described earlier (1.0 ml) were
denatured at 60ꢀC in a water bath for 30 minutes. The resulting precipitated
protein was suspended in 200 ml of ammonium bicarbonate solution (50 mM,
pH 8.0) containing dithiothreitol (5.0 mM), followed by incubation at 60ꢀC for
1 hour. Then, a mixture of Pronase E (2.0 mg/ml), chymotrypsin (1.0 mg/ml), and
CaCl2 (5.0 mM) was added with continuous incubation at 37ꢀC for 12 hours. The
digestion mixtures were centrifuged, and the resulting supernatants were mixed
with 10 ml of IS solution (50 ng/ml of propranolol) before LC-MS/MS analysis.
Materials and Methods
Materials
BBR (.98%) was obtained from Aladdin Industrial Technology Co., Ltd.
(Shanghai, China). Dithiothreitol ($97%), N-acetylcysteine (NAC; .97%),
GSH (.98%), L-cysteine (.97%), and reduced nicotinamide adenine dinucle-
otide phosphate (NADPH) were purchased from Sigma-Aldrich (St. Louis, MO).
Pronase E (.98%) was acquired from Shanghai Yuanye Biological Technology
Co., Ltd. (Shanghai, China). All organic solvents were from Fisher Scientific
(Springfield, NJ). All reagents used for experiments were of either analytical or
high-performance liquid chromatography grade.
Microsomal Incubations
Mouse liver microsomes were prepared as we published previously (Lin et al.,
2007). Incubation mixtures contained mouse liver microsomes (1.0 mg protein/ml);
BBR (75 mM); MgCl2 (3.2 mM); and L-cysteine, NAC, or GSH (10 mM) in
500 ml of phosphate buffer (pH 7.4). The reaction was commenced by addition
of NADPH (1.0 mM) and quenched by addition of equal volume of ice-cold
acetonitrile after 60-minute incubation at 37ꢀC. Control incubations without
NADPH were included. The incubation mixtures were vortex mixed and then
centrifuged at 4ꢀC at 19,000g for 10 minutes to remove precipitated protein.
The resulting supernatants were dried and reconstituted in 100 ml of 50% aceto-
nitrile in water, followed by LC-MS/MS analysis.
Animal Experiments
Male Kunming mice (22 6 2 g) were supplied by the Animal Center of
Shenyang Pharmaceutical University (Shenyang, China). Animals were acclima-
tized at a temperature- and humidity-controlled facility through 12-hour light/dark
cycles and allowed free access to food and water. All procedures were in accordance
with the guide for the Ethics Review Committee for Animal Experimentation of
Shenyang Pharmaceutical University.
One group of mice (n 5 4) treated intraperitoneally with vehicle were
individually placed in metabolic cages overnight for the collection of blank
urine. Then mice were treated (intraperitoneally) with BBR dissolved in
Chemical Synthesis
Mercapturic acid 11 was synthesized as described in Scheme 1 to facilitate
metabolite identification and to define the oxidative debromination pathway
of BBR.
2-Acetylbenzofuran (3). 2-Hydroxybenzaldehyde (1, 0.60 g, 4.91 mmol) was
corn oil (25 mg/ml, 10 ml/kg) at a dose of 100 mg/kg. Urine samples were added to a mixture of KOH (0.33 g, 5.89 mmol) in ethanol (30 ml) with stirring.
collected up to 24 hours after the administration and kept cold on ice. Free The resulting yellow mixture was refluxed for 30 minutes, followed by dropwise
access to water was provided during the experiment.
addition of chloroacetone (2, 0.68 g, 7.37 mmol). The solution was filtered
In a separate study, mice were treated (i.p.) with BBR at doses of 50, 100, immediately after heating under reflux for 3 hours. The solvent was removed
or 200 mg/kg (n 5 4 in each group). The animals were anesthetized, followed under vacuum to yield an orange wet solid (0.71 g, 90% yield). 1H NMR was
by the collection of liver tissues 4 hours after the treatment.
as follows (300 MHz, CDCl3, Supplemental Fig. S4): d 7.70 (doublet or d, 1H,
For time-dependent experiments, mouse livers were harvested at 0.17, 0.5, 1, 2, J 5 5.9 Hz, aromatic-H or Ar-H), 7.57 (doublet of doublets, 1H, J 5 0.5, 6.3 Hz,
4, 8, 12, and 24 hours after the administration of BBR (i.p.) at 100 mg/kg (n 5 4 Ar-H), 7.47 (multiplet or m, 2H, Ar-H), 7.31 (m, 1H, Ar-H), 2.60 (single or s, 3H, CH3).
for each time point). At the time points of 1, 2, and 4 hours, gallbladders were also
13C NMR was as follows (150 MHz, CDCl3, Supplemental Fig. S9): 188.62,
collected for the detection of GSH conjugates. Blank bile collected from vehicle- 155.65, 152.64, 128.24, 127.03, 123.88, 123.26, 112.99, 112.43, 26,42.
treated mice was included. The collected biologic samples were stored at 280ꢀC Mass spectrometry (MS) was as follows [electrospray ionization (ESI)]:
until analysis.
m/z 160.1 [M1H]1.

In Vivo Oxidative Debromination of Benzbromarone
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Scheme 1. Synthetic routes of BBR-NAC
conjugate (11). DMF, dimethyl formamide;
RT, room temperature.
2-Ethylbenzofuran (4). A solution of 2-acetylbenzofuran (3, 0.52 g, 3.22 mmol) allowed to stir at room temperature for 21 hours. The reaction mixture was
and hydrazine monohydrate (0.40 g, 8.05 mmol) suspended in diethylene glycol diluted with water and extracted with CH2Cl2. The pooled organic layer was
(50 ml) was mixed, slowly heated to 150ꢀC, and refluxed for 30 minutes. After washed with HCl (0.5 N), water, NaHCO3, and brine. The resultant CH2Cl2
cooling to room temperature, KOH (0.36 g, 6.44 mmol) was added, and the layer was dried with anhydrous sodium sulfate, filtered, evaporated, and purified
resulting mixture was stirred for 3 hours under refluxing. The solution was next via silica gel column chromatography to give the yellow oil (0.2 g, 30% yield).
diluted with 20 ml of water and extracted with CH2Cl2 (20 ml  3). The combined 1H NMR was as follows (300 MHz, CDCl3, Supplemental Fig. S7): d 7.61 (d, 1H,
organic phase was washed with water and brine, dried with anhydrous sodium J 5 1.44 Hz, Ar-H), 7.46 (m, 2H, Ar-H), 7.39 (d, 1H, J 5 1.41 Hz, Ar-H),
sulfate, and evaporated to yield a brown oil (0.4 g, 85% yield). 1H NMR was 7.29 (m, 1H, Ar-H), 7.23 (m, 1H, Ar-H), 3.94 (s, 3H, OCH3), 3.87 (s, 3H, OCH3),
as follows (300 MHz, CDCl3, Supplemental Fig. S5): d 7.34 (m, 2H, Ar-H), 2.89 (quartet, 2H, J 5 5.67 Hz, CH2), 1.34 (triplet, 3H, J 5 5.64 Hz, CH3).
7.07 (m, 2H, Ar-H), 6.24 (d, 2H, J 5 0.7 Hz, Ar-H), 2.68 (m, 2H, CH2), 13C NMR was as follows (150 MHz, CDCl3, Supplemental Fig. S12):
1.21 (m, 3H, CH3). 13C NMR was as follows (150 MHz, CDCl3, Supplemental
189.40, 166.29, 153.62, 153.59, 150.28, 135.62, 126.71, 126.67, 124.48,
Fig. S10): 160.93, 154.65, 129.00, 123.03, 122.33, 120.15, 110.64, 100.95, 21.75, 123.59, 121.14, 117.28, 115.61, 111.91, 111.00, 60.74, 56.19, 21.91, 12.22. MS
11.82. MS was as follows (ESI): m/z 146.1 [M1H]1.
3-Bromo-4,5-dimethoxybenzoic Acid (6). 3-Bromo-4,5-dimethoxybenzaldehyde
was as follows (ESI): m/z 389.0 [M1H]1 (50%), 391.0 [M1H]1 (50%).
2-Ethyl-3-(3-bromo-4,5-dihydroxybenzoyl)-benzofuran (9). To a solution
(5, 0.65 g, 2.65 mmol) was added to a solution of 0.1 M NaHCO3 (30 ml). The of 2-ethyl-3-(3-bromo-4,5-dimethoxybenzoyl)-benzofuran (8, 0.16 g, 0.41 mmol)
resulting suspension was slowly heated to 90ꢀC. After 10-minute stirring, KMnO4 in CH2Cl2 (10 ml) under a nitrogen atmosphere, precooled at 230ꢀC, 1 M
(0.42 g, 2.65 mmol) was added and the mixture was stirred for 1 hour, followed by BBr3/CH2Cl2 (1.64 ml, 1.64 mmol) was added. The mixture was stirred at
filtration. The resulting filtrates were washed with 10% HCl, and white solid (0.62 g, 230ꢀC for 2 hours, and then stirred at room temperature for 24 hours. The
90% yield) was obtained. 1H NMR was as follows (300 MHz, CDCl3, Supplemental reaction was quenched by addition of cold water (10 ml) and then extracted with
Fig. S6): d 9.84 (broads, 1H, OH), 7.94 (s, 1H, Ar-H), 7.58 (s, 1H, Ar-H), 3.94 (s, 6H, CH2Cl2. The pooled organic layer was washed with water and brine, dried with
2 Â OCH3). 13C NMR was as follows (150 MHz, CDCl3, Supplemental Fig. S11): anhydrous sodium sulfate, and concentrated. The product was purified by silica
170.51, 153.36, 151.21, 127.47, 125.65, 117.51, 113.11, 60.76, 56.26. MS was as gel column chromatography to give a brown solid (0.12 g, 82% yield). 1H NMR
follows (ESI): m/z 260.9 [M1H]1 (50%), 262.9 [M1H]1 (50%).
was as follows (300 MHz, CDCl3, Supplemental Fig. S8): d 10.45 (broads, 2H,
3-Bromo-4,5-dimethoxybenzoyl Chloride (7). A catalytic amount of dry OH), 7.77 (d, 1H, J 5 8.15 Hz, Ar-H), 7.51 (m, 5H, Ar-H), 2.95 (quartet,
dimethyl formamide (one drop) was added to a stirred solution of dried carboxylic 2H, J 5 7.70 Hz, CH2), 1.40 (triplet, 3H, J 5 7.44 Hz, CH3). 13C NMR was as
acid 6 (0.54 g, 2.06 mmol) in 15 ml of SOCl2. The reaction was allowed to reflux follows (150 MHz, CDCl3, Supplemental Fig. S13): 186.36, 162.46, 151.07,
at 78ꢀC for 3 hours and was then cooled to room temperature. The excess of
SOCl2 was removed in a vacuum to give benzoyl chloride 7, which was used
directly for the following Friedel-Crafts reaction.
146.08, 144.07, 128.46, 124.66, 123.06, 122.65, 121.75, 118.80, 113.47, 112.69,
109.17, 107.26, 22.53, 11.97. MS was as follows (ESI): m/z 361.0 [M1H]1 (50%),
363.0 [M1H]1 (50%).
2-Ethyl-3-(3-bromo-4,5-dimethoxybenzoyl)-benzofuran (8). Benzoyl
chloride 7 dissolved in CH2Cl2 was added dropwise to a vigorously stirred solution
of 2-ethyl-benzofuran (4, 0.25 g, 1.72 mmol) in 10 ml of CH2Cl2which was
precooled in an ice-water bath. Next, tin (IV) chloride (0.54 g, 2.06 mmol) was
carefully added into the mixture. The mixture was stirred for 3 hours and then
Mercapturic Acid 11
To a stirred solution of compound 9 (0.1 g, 0.28 mmol) dissolved in 10 ml of
CH2Cl2, 10 ml of NaIO4 (1.2 g, 5.6 mmol) in water was added over 2 minutes, and

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Wang et al.
Fig. 1. LC-MS/MS (m/z 524/336) chromatograms of urine samples obtained from mice before (A) or after (B) treatment with BBR at 100 mg/kg. (C) LC-MS/MS (m/z 524/336)
chromatogram obtained from analysis of microsomal incubations containing NAC as a trapping agent in the presence of NADPH. (D) LC-MS/MS (m/z 522→334) chromatogram
obtained from analysis of synthetic BBR-NAC conjugate (11). (E–G) MS/MS spectra of M1 obtained from analysis of urine of BBR-treated mice, microsomal incubations, or
chemical synthesis.
the resulting biphasic mixture was stirred vigorously at room temperature.
in vacuum to remove ethanol, then extracted with CH2Cl2. The remaining
After 3 hours, compound 9 was completely vanished as monitored by thin CH2Cl2 layer was washed with water and brine, dried with anhydrous
layer chromatography. The biphasic mixture was separated, and the organic sodium sulfate, and concentrated, and the crude product was submitted to
phase was washed with water and concentrated in vacuo to afford the crude semipreparative high-performance liquid chromatography for purification
benzoquinone product as a dark red solid. The solution of the benzoquinone of target compound 11. Unfortunately, the poor yield of compound 11 only
in ethanol (10 ml) was added dropwise into a stirred solution of NAC (0.09 g,
allowed us to characterize it by mass spectrometry, as shown in the Results
0.56 mmol) in 4 ml of water. After 4 hours, the reaction mixture was concentrated section.

In Vivo Oxidative Debromination of Benzbromarone
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Fig. 2. High-resolution mass spectrum of synthetic BBR-NAC conjugate [11 (A)] and BBR standard (B) analyzed by microQ-TOF MS (Bruker Corporation).
LC-MS/MS Method
Analytical separations of NAC conjugate, GSH conjugate, and cysteine adduct
were achieved on an Agilent Poroshell 120 XB-C18 column (4.6 Â 100 mm,
2.7 mm; Agilent Technologies) at a flow rate of 0.5 ml/min. The mobile phases
were composed of 0.1% formic acid in water (A) and 0.1% formic acid in
acetonitrile (B). Ten microliters of sample was injected and separated as
follows: 10% B for 2 minutes, 10%–100% B linear gradient for 12 minutes,
a wash at 100% B for 2 minutes, 100%–10% B linear gradient for 1 minutes,
and re-equilibration at 10% B for 2 minutes (total run time, 19 minutes). For
semipreparative separation of the synthetic NAC conjugate 11, a YMC-Pack
ODS-A column (10 Â 250 mm, S-5, 12 nm; YMC Co., Ltd.) was applied at
a flow rate of 3 ml/min. Separation was accomplished by isocratic elution with
30% B for 50 minutes as indicated earlier.
Sample analyses were carried out using a SCIEX Instrument 5500 triple
quadrupole mass spectrometry (Applied Biosystems, Foster City, CA) connected
to an Agilent 1260 Series Rapid Resolution LC system (Agilent Technol-
ogies, Santa Clara, CA). MS analyses were performed in electrospray
positive ionization mode by multiple reaction monitoring (MRM) scanning.
The MRM transitions (declustering potential, collision energy, collision
cell exit potential), m/z 522→334 (25, 110, 10), m/z 666→391 (30, 110, 10),
m/z 480→334 (25, 110, 10), and m/z 260.3→116.3 (25, 75, 20), were
monitored for NAC conjugate (Fig. 1), GSH conjugate (Figs. 3 and 4),
cysteine adduct (Figs. 5–7), and propranolol (IS), respectively. The optimized
MS conditions were as follows: ion spray voltage (IS), 5500 V; ion source
temperature, 600ꢀC; curtain gas, 35 psi; gas 1 (GS1), 50 psi; GS2, 60 psi.
In addition, a SCIEX Instrument 4000 Q-Trap (Applied Biosystems) equipped
with an Agilent 1260 Series Rapid Resolution LC system was applied to
obtain the MS/MS spectra (Figs. 1, 3, and 5). The information-dependent
acquisition method was used to trigger the enhanced product ion in positive
mode by MRM scanning. The collision energy value was set at 40 eV with
a spread of 5 eV. Other parameters were the same as described earlier except
for GS2 (50 psi) and curtain gas (25 psi). The MS data were analyzed by
Applied Biosystems/SCIEX Analyst 1.6.2 software.
Statistical Analysis
Statistical analyses were performed by unpaired Student’s t test using
GraphPad Prism 5.0 software (GraphPad Software). Statistical significance was
defined as P , 0.05, 0.01, or 0.001.
Results
Urinary NAC Conjugate Derived from Oxidative Debromination
of BBR in Mice. We proposed that oxidative debromination of
BBR in vivo produces o-quinone metabolite 10 (Scheme 2). The
metabolic activation of BBR was first investigated by monitor-
ing urinary excretion of BBR metabolites in mice, using a designed
MRM template as described in the LC-MS/MS Method section. A
metabolite (M1) with retention time at 13.02 minutes (Fig. 1B) was
Mass spectrometry analysis was also performed on a hybrid quadrupole time-
of-flight MS system (microQ-TOF; Bruker Corporation, Billerica, MA) to further
characterize compound 11 (Fig. 2; Table 1). The ESI source was set in negative
mode with the following parameters: capillary voltage, 24,500 V; endplate offset,
2500 V; dry gas, high-purity nitrogen (N2); dry gas flow rate, 4.0 l per minute; gas
temperature, 180ꢀC, and nebulizer gas pressure, 0.3 bar. The data were analyzed
by Bruker Daltonics Data Analyst 3.4 software.
TABLE 1
Summary of mass spectrometric data obtained from microQ-TOF MS analysis of synthetic BBR-NAC conjugate (11) and BBR standard
Description
Formula
Calculated Mass
Measured Mass
Absolute Error
Relative Error
[M-H]2
mDa
ppm
M1
C22H19BrNO7S
520.0071/522.0228
520.0060/522.0211
1.13/1.71
2.52/3.28
BBR
C
17H11Br2O3
420.9067/422.9049/424.9032
420.9047/422.9050/424.9014
2.01/0.13/1.84
4.78/0.31/4.33

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Wang et al.
Scheme 2. Proposed pathways of mercapturic acid and protein adduct formation as a result of ipso-substitution of BBR in vivo. P450, cytochrome P450.
observed in urine samples extracted with solid phase extraction. No characteristic fragment ions, including m/z 591 (loss of 75 Da, glycinyl
such metabolite was observed in blank urine (Fig. 1A). portion) and 537 (loss of 129 Da, glutamyl portion), indicating that GSH
As shown in Fig. 1D, M1 had its protonated molecular ions at participated in the formation of M2. Product ion m/z 519 originated from
m/z 522 (50%) and 524 (50%), signaling that the metabolite was the loss of 129 Da and an H2O. The ions at m/z 391 and 247 were
derived from the loss of a bromine atom from BBR. The MS/MS considered to be the indicative fragmentations of BBR moiety, along
spectrum of M1 provided fragment ions at m/z 334 (indicative with the cleavage of the GSH moiety. The fragment ion at m/z 173
characteristic fragmentation associated with the cleavage of the NAC corresponded to the benzofuran ring of BBR, which suggests that the
moiety) and m/z 391 (a fragment resulting from the cleavage of the addition of GSH took place on the monobrominated benzoyl ring.
C-S bond within NAC), suggesting that NAC participated in the forma- As expected, M2 was observed in GSH-supplemented mouse liver
tion of M1. The fragment ions at m/z 145 and 173 (loss of C10H9O and microsomal incubations of BBR (Fig. 3C), which showed the same
C11H9O2) were found to be the same as the spectrometric pattern of mass spectrometric pattern (Fig. 3E) as the metabolite obtained in mouse
parent BBR (Wang et al., 2016b), suggesting that the benzofuran ring bile (Fig. 3D). The GSH conjugate detected in vivo further indicates the
remained unchanged. This led us to propose that M1 was derived from proposed oxidative debromination of BBR (Scheme 2).
quinone metabolite 10 (a debromination metabolite of BBR) with
GSH conjugate derived from metabolite 10 of BBR was also deter-
NAC attaching to the brominated benzoyl ring. Additionally, M1 was mined in the livers of mice to ensure the proposed oxidative debromi-
observed in NAC-fortified microsomal incubations of BBR (Fig. 1C), nation pathway. As expected, one peak with the same retention time
which shared the same mass spectrometric pattern (Fig. 1E) as that as M2 (found in microsomal incubations; Fig. 3C) was detected by
detected in the urine of BBR-treated mice (Fig. 1D).
LC-MS/MS using the designed MRM template (data not shown).
LC-MS/MS Analysis of Synthetic BBR-NAC Conjugate (M1). Additionally, the time course of GSH conjugate was determined in
To verify the structure of M1, we chemically synthesized NAC conjugate the livers of mice treated with BBR. The GSH conjugate reached its
11 by oxidation of compound 9 to quinone 10, followed by reaction peak 2 hours after administration (Fig. 4).
with NAC (Scheme 1). The synthetic NAC conjugate showed the
Protein Adduction by Oxidative Debromination of BBR in Mice.
same chromatographic behavior (Fig. 1D) and mass spectral iden- Protein modification derived from the debromination metabolite of
tities (Fig. 1G) as those of the metabolite excreted in mouse urine BBR was examined in mice by monitoring the corresponding protein
(Fig. 1, B and E). The product was further analyzed by high-resolution adduction that occurs at cysteine residues. One product (M93) with an
electrospray ionization mass spectrometry in negative mode (Fig. 2). elution time of 11.40 minutes was detected in the liver homogenate
The final product clearly possessed the bromine isotope pattern with after proteolytic digestion (Fig. 5B). No such product was observed
a molecular cluster of m/z 520.0060 (50%) and 522.0211 (50%), and in the digested samples obtained from mice treated with vehicle
the relative errors between the observed and calculated mass were (Fig. 5A).
within 5 ppm (Table 1). Identification of the NAC conjugate provided
important evidence for the oxidative debromination pathway of BBR at m/z 480 (Fig. 5D), which was equal to the calculated m/z value of
in mice. a cysteine conjugate derived from oxidative debromination metabolite
The mass spectrum of M93 displayed its protonated molecular ion
GSH Conjugate Derived from Oxidative Debromination of BBR 10 (Scheme 2). M93 exhibited its major product ion at m/z 334, which
in Mice. In vivo ipso-substitution (oxidative debromination; Scheme 2) was formed by the fracture of the benzoyl moiety of BBR containing an
metabolism of BBR was also investigated by monitoring the GSH intact cysteine. Ion m/z 391 was assigned to be the fragment resulting
conjugate of BBR in the bile of mice. After intraperitoneal injec- from the break of the C-S bond within the cysteine moiety. Product ions
tion of BBR in mice, one glutathione conjugate—namely, M2—was at m/z 247 and 219 were yielded by the cleavages of the benzoyl moiety
detected in bile samples. The corresponding LC-MS/MS chromatogram of BBR, along with the break of the C-S bond within the cysteine
is shown in Fig. 3B. No such metabolite was found in blank bile moiety. Ion m/z 173, which corresponds to the benzofuran ring of BBR,
(Fig. 3A). The observed molecular ion value of M2 was consistent was also observed in the mass spectrum of M93. The MS/MS pattern led
with the calculated m/z value of a GSH conjugate derived from us to surmise that the formation of M93 results from a cysteine
quinone metabolite 10 (oxidative debromination metabolite; Scheme 2). conjugate with quinone metabolite 10. Cysteine- and BBR-fortified
As shown in Fig. 3D, the MS/MS spectrum of M2 showed two microsomal incubations were performed to characterize the structure

In Vivo Oxidative Debromination of Benzbromarone
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Fig. 3. LC-MS/MS (m/z 666→391) chromatograms of bile samples obtained from mice before (A) or after (B) treatment with BBR at 100 mg/kg. (C) LC-MS/MS
(m/z 666→391) chromatogram obtained from analysis of microsomal incubations containing GSH as a trapping agent in the presence of NADPH. (D and E) MS/MS spectra
of M2 obtained from analysis of bile samples of BBR-treated mice or microsomal incubations.
of M93. The cysteine conjugate formed in the incubations shared the
GSH conjugation is considered to be a common detoxifica-
same chromatographic (Fig. 5C) and mass spectrometric identities tion process during the metabolism of xenobiotics. GSH conjugates
(Fig. 5E) as the product generated in the protein digestion.
undergo a further process and are often converted to mercapturic acids
Furthermore, we examined the time course of hepatic protein adduction
in BBR-treated mice over 24 hours. The protein adduction arising from
the debromination metabolite of BBR (10; Scheme 2) reached its peak
4 hours after the administration (Fig. 6). In addition, dose-dependent
hepatic protein adduction was monitored in mice 4 hours after BBR
treatment (50, 100, or 200 mg/kg). The observed protein adduction
increased with the elevation of the dose of BBR given in mice (Fig. 7).
Discussion
The hepatotoxicity of BBR has been well documented, and
cytochrome P450–mediated metabolic activation has been suggested
to be involved in BBR-induced liver injuries. The present study focused
on the investigation of the oxidative debromination pathway of
BBR in vivo, along with protein modification derived from the
resulting reactive metabolite.
Fig. 4. Time-course changes in GSH conjugate. Mice were treated (i.p.) with BBR at
100 mg/kg. GSH conjugate was examined at various time points after administration (n 5 4).
The most abundant GSH conjugate detected in mouse liver tissues was normalized to 100%.

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Wang et al.
Fig. 5. LC-MS/MS (m/z 480→334) chromatograms of liver homogenates obtained from mice before (A) or after (B) treatment (i.p.) with BBR at 100 mg/kg. (C) LC-MS/MS
(m/z 480→334) chromatogram obtained from analysis of microsomal incubations containing cysteine as a trapping agent in the presence of NADPH. (D and E) MS/MS
spectra of M93 obtained from analysis of liver homogenates of BBR-treated mice or microsomal incubations after proteolytic digestion.
(NAC conjugates), which are generally considered as biomarkers of synthesis and messy impurities of the final crude mixture, LC-MS/MS
exposure to electrophiles (Poon et al., 2001; Cosnier et al., 2013). In analysis (Fig. 1, D and G) as well as high-resolution electrospray
vivo studies were initiated from the detection of urinary mercapturic ionization mass spectrometry (Fig. 2; Table 1) data provided strong
acids and biliary GSH conjugates in mice. One NAC conjugate (M1; evidence for the identity of M1. Two GSH conjugates or NAC
Fig. 1B) and one GSH conjugate (M2; Fig. 3B) were detected in the conjugates (Supplemental Fig. S3) derived from quinone metabolite 10
urine and bile samples of BBR-treated mice, respectively. In addition, were expected to be formed, but only one conjugate (M1 or M2) was
these conjugates were also found in microsomal incubations supple- detected in both in vitro and in vivo studies. The failure to form the
mented with NAC or GSH as a trapping agent in an NADPH-dependent second conjugate was proposed to be related to the steric hindrance
manner, and they elicited the same chromatographic and mass spectral of the ethyl side chain.
identities as those found in biologic samples. The product ion spectra
Hepatic protein modification by electrophilically reactive metabolites
showed that M1 and M2 arose from oxidative debromination of BBR, is thought to be an important mechanism for the observed toxicities of
resulting in the formation of a reactive quinone species, which subsequently many xenobiotics (Boysen and Hecht, 2003; Moghe et al., 2015; Yang
reacted with NAC or GSH. The chemical synthesis of the NAC and Bartlett, 2016). One protein adduct (M93) derived from oxidative
conjugate further allowed us to identify M1. Although there has debromination of BBR was observed in BBR-treated mice (Fig. 5B).
been difficulty obtaining a sufficient amount of pure NAC conjugate And such adduction occurred in a dose- and time-dependent manner
(11; Scheme 1) for NMR characterization, owing to the poor yield of the (Figs. 6 and 7). The dosage used in this study was based on our previous

In Vivo Oxidative Debromination of Benzbromarone
1289
Fig. 6. Time-course changes in protein adduction that occurs at cysteine residues.
Mice were treated (i.p.) with BBR at 100 mg/kg. Protein adduction was examined
at various time points after administration (n 5 4). The most abundant protein
adduction detected in mouse liver tissues was normalized to 100%.
Fig. 7. Dose dependency of protein adduction that takes place at cysteine residues.
Mice were treated (i.p.) with BBR at 50, 100, and 200 mg/kg. Protein adduction was
examined 4 hours after administration (n 5 4).
evidence that quinones are linked to a variety of adverse events,
including acute cytotoxicity, hepatotoxicity, and carcinogenesis (Bolton
et al., 2000; Hughes and Swamidass, 2017).
study (Wang et al., 2016a) in which 30, 50, and 100 mg/kg were applied
for the detection of protein adduction derived from the epoxide metab-
olite of BBR. In the present study, we increased the dosage to 50, 100,
and 200 mg/kg, due to the limited sensitivity of mass spectrometry
to detect low levels of protein adducts derived from ipso-substitution
of BBR. The protein adduction reached its peak at 4 hours after
the administration, which was 2 hours later than the peak time for the
formation of the GSH conjugate (Fig. 4). This perhaps results from the
readiness of the electrophilic species to access to the sulfhydryl group
of abundant GSH, while thiol groups of protein may not be that acces-
sible. In an attempt to compare the time courses of the metabolites
of BBR through ipso-substitution and epoxidation pathways, which we
published recently (Wang et al., 2016a,b), the formation of epoxide-
derived GSH conjugate and protein adduction were also monitored
in BBR-treated mice at various time points. The formation of epoxide-
derived GSH conjugate reached its peak (30 minutes) much faster than
did the oxidative debrominated metabolite (2 hours) (Supplemental
Fig. S1). Similarly, the epoxide-derived protein adduction reached its
peak 3.5 hours earlier than the binding resulting from metabolite 10
(Supplemental Fig. S2). This indicates that BBR was more liable to be
transformed into an epoxide intermediate in vivo than the oxidative
debrominated metabolite.
In this study, we investigated the oxidative debromination path-
way of BBR in vivo (Scheme 2). BBR was metabolized to a quinone
intermediate, which reacted with GSH. The GSH conjugate underwent
sequential hydrolysis and acetylation to a mercapturic acid and was
excreted in urine. Protein adduction that happens at cysteine residues
was detected in BBR-treated mice after proteolysis of liver homogenates
with proteinase. The in vivo studies allowed us to better understand
the metabolic activation of BBR, and facilitate the further investigation
of the mechanisms of BBR-induced hepatotoxicity.
Authorship Contributions
Participated in research design: Zheng, Peng.
Conducted experiments: H. Wang, W. Wang, Gong, Z. Wang.
Contributed new reagents or analytic tools: Zhang, S. Wang.
Performed data analysis: H. Wang, Feng.
Wrote or contributed to the writing of the manuscript: H. Wang, Zheng.
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Address correspondence to: Ying Peng, Wuya College of Innovation,
Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, P. R.
China. E-mail: yingpeng1999@163.com; or Jiang Zheng, State Key Labora-
tory of Functions and Applications of Medicinal Plants, Key Laboratory of
Pharmaceutics of Guizhou Province, Guizhou Medical University, Guiyang,
Guizhou 550004, P. R. China.; Wuya College of Innovation, Shenyang
Pharmaceutical University, 103 Wenhua Road, Shenyang, Liaoning 110016,
P. R. China. E-mail: zhengneu@yahoo.com
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