Chemical Reactivity of Aloe-Emodin and Its Hydroxylation Metabolites to Thiols

Article abstract of DOI:10.1021/acs.chemrestox.8b00248

Aloe-emodin (AE), an anthraquinone derivative, is a bioactive ingredient isolated from rhubarb which is used to treat inflammatory illnesses in China and many other countries in Asia. AE has shown a wide range of pharmacological effects. Recent studies showed that exposure to AE could cause DNA damage and cytotoxicity. The goals of the present study are aimed at (1) exploration of oxidative metabolism pathways of AE, (2) identification of P450 enzymes which respond the hydroxylation of AE, and (3) determination of electrophilicity of AE and its oxidative metabolites. Two hydroxylation metabolites (M1 and M2) and four GSH conjugates (M3-M6) were found in incubations consisting of AE, rat or human liver microsomes, and NADPH supplemented with GSH. Conjugates M3 and M4 came from AE itself, and M5 and M6 originated from M1 and M2 individually. M1 and M2 (5-hydroxy aloe-emodin) and M3-M6 were also detected in rat primary hepatocytes after exposure to AE. Additionally, biliary M3, M4, and M6 were detected in rats given AE. Urinary M1, M2, and M7 (a NAC conjugate) were observed in animals administered AE. Recombinant P450 enzyme incubations illustrated that hydroxylation of AE was primarily catalyzed by P450 1A2, 3A4, and 3A5. The metabolism investigation will help us to better understand the biochemical mechanisms of cytotoxicity induced by AE.

Full text of DOI:10.1021/acs.chemrestox.8b00248

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Article
Chemical Reactivity of Aloe-Emodin and
Its Hydroxylation Metabolites to Thiols
Xu Wang, Xin Xin, Ying Sun, Lizhu Zou, Hui Li, Yufei Zhao, Ruihong Li, Ying Peng, and Jiang Zheng
Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00248 • Publication Date (Web): 23 Jan 2019
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Chemical Reactivity of Aloe-Emodin and Its Hydroxylation Metabolites to Thiols
Xu Wang,¶,† Xin Xin,¶,† Ying Sun,† Lizhu Zou,† Hui Li,† Yufei Zhao,† Ruihong Li,†
Ying Peng,*,† and Jiang Zheng*,†,§
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†Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang,
Liaoning, 110016, P.R. China
§State Key Laboratory of Functions and Applications of Medicinal Plants, Key
Laboratory of Pharmaceutics of Guizhou Province, Guizhou Medical University,
Guiyang, Guizhou, 550025, P.R. China
*Corresponding Authors:
Jiang Zheng, PhD
Wuya College of Innovation, Shenyang Pharmaceutical University, No. 103, Wenhua
Road, Shenyang, Liaoning, 110016, P. R. China
State Key Laboratory of Functions and Applications of Medicinal Plants, Key
Laboratory of Pharmaceutics of Guizhou Province, Guizhou Medical University, 9
Beijing Road, Guiyang, Guizhou, 550025, P.R. China
Email: zhengneu@yahoo.com
Tel: +86-24-23986361; Fax: +86-24-23986510
Ying Peng, PhD
Wuya College of Innovation, Shenyang Pharmaceutical University, No. 103, Wenhua
Road, Shenyang, Liaoning, 110016, P. R. China
Email: yingpeng1999@163.com
Tel: +86-24-23986361; Fax: +86-24-23986510
¶: The two authors contributed equally to this work.
†, §: The two corresponding units contributed equally to this work.
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ABSTRACT
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Aloe-emodin (AE), an anthraquinone derivative, is a bioactive ingredient
isolated from Rhubarb which is used to treat inflammatory illnesses in China and
many other countries in Asia. AE has shown a wide range of pharmacological
effects. Recent studies showed that exposure to AE could cause DNA damage and
cytotoxicity. The goals of the present study aimed at 1) exploration of oxidative
metabolism pathways of AE, 2) identification of P450 enzymes which respond the
hydroxylation of AE, and 3) determination of electrophilicity of AE and its oxidative
metabolites. Two hydroxylation metabolites (M1 and M2) and four GSH conjugates
(M3-M6) were found in incubations consisting of AE, rat or human liver microsomes,
and NADPH supplemented with GSH. Conjugates M3 and M4 came from AE itself,
and M5 and M6 originated from M1 and M2 individually. M1 and M2 (5-hydroxy
aloe-emodin) and M3-M6 were also detected in rat primary hepatocytes after
exposure to AE. Additionally, biliary M3, M4 and M6 were detected in rats given
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AE.
Urinary M1, M2 and M7 (a NAC conjugate) were observed in animals
Recombinant P450 enzyme incubations illustrated that
administered AE.
hydroxylation of AE was primarily catalyzed by P450 1A2, 3A4 and 3A5. The
metabolism investigation will help us to better understand the biochemical
mechanisms of cytotoxicity induced by AE.
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INTRODUCTION
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Rhubarb (Rheum rhabarbarum), a famous herb known as traditional Chinese
medicine, is widely utilized for the treatment of many diseases, such as constipation,
jaundice, and gastrointestinal bleeding and ulcers.1 Rhubarb is often employed in
many Chinese herbal medicine formulations applied for the treatment of inflammation,
including acute cholecystitis, rheumatoid arthritis and acute appendicitis.2 Rhubarb
also reportedly showed anti-bacterial, antioxidant, anti-cancer, anti-angiogenesis and
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anti-inflammation properties.3-6
Aloe-emodin (AE) is one of major natural
anthraquinone ingredients in Rhubarb.7 Recently, AE has caught much attention
because of its various pharmacologic properties, including cardiovascular protection,
laxative, antifungal, antiviral, skin conditioning, and immune regulation effects.8
According to recent reports, AE revealed selective inhibitory effect on the growth of
neuroectodermal tumors.9 Meanwhile, AE was reported to strengthen anticancer
effect when co-administered with cisplatin, doxorubicin, docetaxel, and
5-fluorouracil.10 In addition, AE enhanced chemotherapeutic efficacy of anticancer
agents (doxorubicin, cis-platinol, 5-fluorouracil, and the tyrosine kinase inhibitor STI
571) in Merkel skin carcinoma cells,11 suppressed the growth of NCI-H460 (human
lung cancer cells)12 and the proliferation of MKN45 gastric cancer cells.10
Interference of DNA functioning, such as DNA damage, is one of the most
common mechanisms of antitumor action. Camptothecin is a topoisomerase I (Top1)
inducer, which induces Top1-mediated DNA damage resulting in reduced cell
proliferation and survival.13,14
Similarly, anthraquinone anticancer drugs
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demonstrated cytotoxic activities through their interaction with guanine/cytosine rich
sites of DNA preferentially.15 The suppression of DNA replication is thought to
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result from certain conformational changes in the DNA.16
Furthermore,
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anthroquinones were reported to produce topoisomerase II-mediated DNA damage.15
AE was found to induce primary DNA damage in the liver and even in the kidney,
which indicates a genotoxic mechanism of action involved in vivo.17 In addition,
micronucleus assay displayed genotoxicity induced by AE itself and its metabolites in
Salmonella typhimurium strains TA98 and TA1537.17 A recent study showed that
exposure to AE and UV light (320-400 nm) induced phototoxicity in cultured human
skin fibroblast cells.18 Moreover, Strickland et al. reported the enhancement UV
light-induced malignant melanoma in C3H mice after topical treatment of AE.19
In this study, we defined the oxidative metabolism pathways of AE in liver
microsomal incubations, cultured primary rat hepatocytes and rats, identified the P450
primary enzymes which participated in the oxidation of AE, and determined the
chemical reactivity of AE and its hydroxylation metabolites towards biological thiols.
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EXPERIMENTAL PROCEDURES
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Chemicals and Materials.
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AE (≥ 98%) was provided by Chengdu Pufeide Biologic Technology Co., Ltd
(Chengdu, China). Human liver microsomes and recombinant human P450 enzymes
were acquired from BD Gentest (Woburn, MA). Rat (Sprague-Dawley, male) liver
microsomes were pre-prepared in our laboratory in accordance with previous
literature.20 Distilled water and organic solvent were obtained from Wahaha Co.,
Ltd. (Hangzhou, China) and Fisher Scientific (Springfield, NJ), respectively.
Glutathione (GSH), S-hexylglutathione, N-acetylcysteine (NAC), and reduced
nicotinamide adenine dinucleotide phosphate (NADPH) were purchased from
Sigma-Aldrich (St. Louis, MO).
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Microsomal Incubation.
AE (27 mg) was dissolved in 10 mL dimethyl sulfoxide (DMSO) to offer the
stock solution. Test compound AE (100 μM) was mixed with rat or human liver
microsomes (1.0 mg protein/mL) in PBS buffer (pH 7.4) and MgCl2 (3.2 mM)
fortified with GSH (10 mM) (final volume: 250 μL, final organic solvent content:
0.5%). The microsomal solution was further mixed with NADPH (1.0 mM) to
initiate the reactions.
Control groups excluded NADPH.
Incubation of the
resulting mixtures was carried out in a water bath shaker. After incubation at 37 °C
for 45 min, the reactions were terminated by addition of the same volume of cold
CH3CN (-20 °C).
Deproteinization was achieved by 10 min centrifugation at
19,000g at 4 C, and the supernatants were analyzed by LC-MS/MS.
Each
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incubation was repeated twice. Similar microsomal incubations were carried out
except the absence of GSH.
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Cell Experiments.
All animal operation procedures were authorized by the Ethics Review
Committee for Animal Experimentation of Shenyang Pharmaceutical University
(license number: SYPU-IACUC-C2018-6-8-201).
Male Sprague-Dawley rats
(200±20 g) were obtained from the Animal Center of Shenyang Pharmaceutical
University. Rats were kept on standard rat chow at room temperature and moderate
humidity with 12 h dark/light cycles. Animals were starved for 12 h and were
anesthetized by use of 10% chloral hydrate (35 mg/kg). Subsequently, rats (except
the head) were soaked in 75% ethanol for 10 s. The rats were fixed, and the
abdomen was opened to expose and isolate the portal vein. An intravenous syringe
(detaining needle) was inserted into the portal vein. The liver was perfused with
perfusion fluid containing 1,000 U/mL heparin. At the same time, the inferior vena
cava was cut in order to discharge the perfusate. When the color of the liver was
changed from light red to pale yellow, the liver was separated and transferred to
sterile petri dishes.
The liver was perfused with 0.05% calcium-containing
collagenase IV perfusate at 37 C at the speed of 5 mL/min until the liver became soft
and fragile. The liver was transferred to a pre-cooled medium containing DMEM.
The hepatocyte suspensions were filtered through a 100 mesh filter to get rid of
connective tissues and then centrifuged at 300 rpm (3 min) and 4 C.
The
supernatants were discarded, and the resulting cell pellets were suspended in a density
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separation solution, followed by 5 min centrifugation at 500 rpm and 4 C.21 The
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supernatants were discarded, and the resulting hepatocyte pellets were placed in
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culture media containing 20% FBS.
The number and viability of cells were
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measured by trypan blue exclusion. Only cells with a viability > 80% were used for
metabolism tests. Cells were incubated in media containing insulin (0.5 mg/L),
dexamethasone (1.0 M) and double antibody (1%). The cell density was 1×105/mL.
All operations were carried out under aseptic conditions.
Cells were incubated with AE and vehicle DMSO for 12 h under 5% CO2 at 37
C in an incubator. The resultant cell suspension was mixed with three volumes of
cold CH3CN (-20 °C) for deproteinization. The resulting supernatants (10 L) were
analyzed by an LC-MS/MS system.
Chemical Synthesis of AE Metabolites.
Chemical synthesis of M1 and M2 were performed in accordance with a
published protocol.22 In short, potassium persulfate (208 mg) was slowly added into
a sulfuric acid solution (4 mL) containing AE (80 mg) with stirring. The resulting
solution was further stirred for another 15 min at room temperature, followed by
mixing with an excess of potassium persulfate (208 mg), stirring at 50 °C for 1 h, and
mixing with 80 mL water and sodium metabisulfite (800 mg).
The resultant
suspension was extracted with butanol (3×4 mL). The butanol extracts were pooled,
washed with dilute hydrochloric acid (3×12 mL) and water (3×12 mL), dried with
anhydrous Na2SO4, and concentrated by vacuum evaporation. A semi preparative
HPLC system was employed for product purification. The purified products were
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characterized by MS and NMR.
All NMR spectra were acquired on a
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BRUKER-ARX-600 spectrometer (600 MHz).
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Synthesis of GSH conjugates M3 and M4 was achieved by mixing of AE (0.05
mol) with GSH (10 mol) in PBS buffer (0.5 mL, pH 7.4), and the resulting solution
was stirred for 1 h at 37 °C and centrifuged at 19,000 g for 10 min. The resultant
supernatants were subjected to the LC-MS/MS system for analysis.
Synthetic GSH conjugates M5 and M6 were prepared as following: M1 (3.48
mol) and M2 (3.48 mol) were separately dissolved in 1.0 mL of methanol, and then
mixed with GSH (20 mM) in 10 mL PBS buffer (pH 7.4). After stirring at 40 °C for
1 h, the resulting mixture was centrifuged at 19,000 g for 10 min and analyzed by an
LC-MS/MS system.
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Animal Experiments
Male Sprague−Dawley rats (200±20 g) were purchased from the Animal
Center of Shenyang Pharmaceutical University were kept on standard rat chow at
room temperature and moderate humidity with 12 h dark/light cycles. Animals were
randomly split into two groups, and each group contained 3 rats. The animals were
starved for 24 h before any treatment. Rats of group one were administered chloral
hydrate (10%, 3.0 mL/kg, i.p.), and bile ducts of the anaesthetized rats were
cannulated, followed by collection of blank bile. Subsequently, the cannulated rats
were intraperitoneally given corn oil solution of AE (200 mg/kg), and then 0–12 h
bile samples were harvested. Rats of the other group were treated with the same
dose of AE (i.p.) and kept in metabolism cages individually.
The rats were
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permitted to reach food and water during the experiment, and 0-24 h urine samples
were collected, along with blank urine samples before the administration of AE.
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Recombinant Human P450 Incubations.
Similar procedure was followed for recombinant P450 incubations as for that
of microsomal reactions described above. The individual human P450 enzymes (100
nM) included P450s 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5. The
final reaction volume was 250 L.
The resulting enzymatic reactions were
terminated by mixing with 250 L cold CH3CN (-20°C) containing
S-hexylglutathione (the internal standard, 50 ng/mL), followed by 30 s vortex mixing
and 10 min centrifuging at 19,000 g. The resultant supernatants were submitted to
an LC-MS/MS system for analysis as below. The generation of the corresponding
products was monitored and regarded as individual enzyme activity.
Normalization
of reaction rates for individual P450 enzymes (NRR) was achieved by the following
equation.23
NRR = RMP × MSC
Where RMP: the rates of metabolite production in incubations with the
individual recombinant P450 enzymes; MSC: the mean specific content of the
corresponding P450 enzyme in human liver microsomes.
Sample Preparation for LC-MS/MS Analysis
Cleaning of bile and urine samples was achieved on C18 SPE cartridges. Bile
and urine samples (pH=3-4, 200 L) were individually loaded onto SPE cartridges
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which had been washed with methanol and then diluted acetic acid (pH 3-4). The
resulting cartridges were washed with deionized water (pH 3-4, acidified with acetic
acid), followed by eluting with a series of gradient methanol solutions (10, 20, 30, 40,
50, 60, 70, 80 and 90%). The resulting eluates were concentrated by blowing with
nitrogen gas, and the residues were dissolved with 10% CH3CN in water (200 μL).
After 10 min centrifuging at 16,000 g and 4 C, and the resulting supernatants (10 μL)
were submitted to the LC-MS/MS for analysis.
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LC-MS/MS Method.
All separations and analyses of samples were conducted on an LC-MS/MS
composed of a 1260 infinity LC system (Agilent Technologies, Biblingen, Germany)
and an inline AB SCIEX Instruments 5500 triple quadrupole mass spectrometer
(Applied Biosystems, Foster City, CA, USA). An Accuore C18 column (4.6×150
mm, 5μm; Thermo Fisher, Inc., Pittsburgh, PA) was employed for analyte separation
with flow rate of 0.8 mL/min and column temperature of 25 C. Solvent A was
CH3CN with 0.1% formic acid and solvent B was water with 0.1% formic acid. The
gradient elution program was set as follows, 0-2 min: 10% solvent A, 2-10 min:
10%-95% A, 10-15 min: 95% A, 15-16 min: 90%-10% A, 16-18 min: 10% solvent A.
The parameters of mass spectrometric analyses were set after optimization as follows,
gas 1: 50 psi, gas 2: 50 psi, curtain gas: 35 psi, ion source temperature (TEM): 650 C,
ion spray voltage (IS): 5,500, declustering potential (DP): 110 V, entrance potential
(EP): 10 V, collision cell exit potential (CXP): 13 V.
Detection of AE,
hydroxylation metabolites of AE, GSH conjugates, and NAC conjugate was achieve
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by monitoring precursor/product ion pairs (collision energy, CE) m/z 271→225 (35),
m/z 287 → 241 (35), m/z 576 → 301 (42)/592 → 317 (42), and 448 → 317 (40),
respectively. Analysis of S-hexylglutathione was performed by scanning ion pair
(DP, CE) m/z 392→246 (86, 24).
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Additionally, an AB SCIEX Instruments 4000 Q-Trap mass spectrometry
equipped with a 1260 infinity LC system was used for enhanced product ion (EPI)
analysis which allowed us to accumulate mass spectral information for all
metabolites/products interested. The information-dependent acquisition (IDA) was
utilized to launch EPI spectrum when ions acquired via multiple reaction monitoring
(MRM) reached over 1,000 cps. The collision energy (CE) was 40 eV with an
extendibility of 15 eV. Positive mode was employed for EPI acquisition by scanning
product ions at a range of m/z 50 to 600. AB SCIEX Analyst 1.6.2 software was
employed to process mass spectrometric data.
Analysis of synthetic metabolites was also carried out on a hybrid
quadrupole-time-of-flight (Q-TOF) mass system (micro Q-TOF, Bruker Corporation,
Billerica, MA) equipped with an inline Agilent 1200 Series Rapid Resolution LC
system.
Analysis was performed in positive-ion mode.
Mass spectrometric
parameters were optimized as follows. Capillary voltage: -4,500 V, end plate offset:
2,500 V, nebulizer gas pressure: 1.2 bar, temperature: 180 °C, the dry gas flow rate:
8.0 L/min. The spectra were obtained at 2 s per spectrum ranging m/z 50 to 1,500.
Similar LC parameters were applied for analyte separation as described above.
Bruker Daltonics Data Analysis 3.4 software was employed to process mass
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spectrometric data.
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RESULTS
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Mass Spectrometric Property of AE.
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Mass spectral behavior of AE was determined to gain major fragments of the
parent compound. Ionization in positive mode was found to provide more fragment
ions and much higher sensitivity than that in negative ionization mode. As shown in
Figure 1, AE displayed m/z 115 ([M+H−H2O−4CO−C2H2]+), 141
([M+H−H2O−4CO]+), 169 ([M+H−H2O−3CO]+), 197 ([M+H−H2O−2CO]+), and 225
([M+H−H2O−CO]+) of fragment ions with molecular ion of m/z 271.
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Formation of Oxidative Metabolites
Liver microsomal incubations (both rat and human) of AE produced two
oxidative metabolites (M1 and M2) with their retention time at 9.53 and 12.0 min
(Figure 2B).
NADPH was required for the production of the two oxidative
metabolites (Figure 2A). This suggests the involvement of P450 enzymes in the
generation of M1 and M2.
The observed [M]+ of M1 and M2 allowed us to consider that the formation of
the metabolites resulted from hydroxylation, and that they are positional isomers
arising from the location of hydroxyl group. The tandem mass spectra of M1 and
M2 displayed product ions m/z 139 ([M+H−2H2O−4CO]+), 157 ([M+H−H2O−4CO]+),
185 ([M+H−H2O−3CO]+), 213 ([M+H−H2O−2CO]+), and 241 ([M+H−H2O−CO]+)
obtained from MRM-EPI scanning (m/z 287/241) (Figure 2E, F). The fragment ions
at m/z 157, 185, 241 and 257 of M1 and M2 were 16 Da higher than ions m/z 141, 169,
225 and 241 of AE (Figure 1), which suggests that M1 and M2 were the products of
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metabolic hydroxylation of AE. Traces of M1 and M2 were found in the authentic
sample, and the observed the contaminated M1 and M2 may result from
auto-oxidation.
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Chemical synthesis of M1 and M2 was achieved by oxidation AE with
potassium persulfate in H2SO4. The resulting products displayed identical retention
time and mass spectral behaviors as M1 and M2 generated in microsomal reactions
(Figure 2C, D, G, and H). High resolution mass spectrometry analysis of synthetic
M1 revealed its protonated molecular ion of m/z 287.0550, consistent with the
corresponding theoretical mass within 5 ppm compared with the predicted formula
(Table 1). Unfortunately, we were not able to gain satisfactory NMR spectrum of
M1. Synthetic M2 was purified and analyzed by NMR for characterization. 1H
NMR (CDCl3, 600 MHz): δ4.85 (2H, S, H-11), 7.31 (1H, d, J=9.4 Hz, H-7), 7.33
(1H, d, J=9.4 Hz, H-6), 7.37 (1H, br. s, H-2), 7.87 (1H, br. s, H-4), 12.22 (1H, s,
1-OH), 12.32 (1H, s, 8-OH), 13.02 (1H, s, 5-OH). M2 was assigned to be 5-hydroxy
aloe-emodin.
Formation of GSH Conjugates.
Four GSH conjugates (M3-M6) were detected in AE-fortified human or rat
liver microsomes supplemented with GSH (Figures S2 and 3). M3 and M4 with
identical molecular ion of m/z 576 eluted at 7.95 and 8.30 min (Figure 3B). In
addition, the mass spectra demonstrated characteristic product ions related to the
cleavage of GSH portion (Figure 3D, E). Primary product ion m/z 301 arose from
the segmentation of the C-S of the GSH portion (−273 Da) and the loss of two
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hydrogen atoms from the GSH conjugates (m/z 576). Fragment ions m/z 501 and
430 resulted from the GSH conjugates (m/z 576) after losing the glycinyl portion (−75
Da) and glutaminyl portion (−146 Da). Product ion m/z 327 came from the loss of
the N-formylglycinyl section (−103 Da) from m/z 430. Meanwhile, both M3 and M4
were found in NADPH-absent microsomal incubations (Figure 3A). Mixing and
reaction of GSH and AE in buffer offered two GSH conjugates whose
chromatographic and mass spectrometric identities (Figure 3C, F and G) are
consistent with those of M3 and M4 formed in microsomal incubations. However,
owing to poor yields of the reactions, we failed to gain sufficient quantities of the two
compounds to be characterized by NMR.
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The formation of M5 and M6 (retention time = 7.73 and 8.16 min) was
monitored by acquiring precursor/product ion pair m/z 592→317 (Figure 4B). The
observed molecular ions of m/z 592 corresponded to the molecular weight of the
conjugates composed of a molecule of GSH and a molecule of hydroxylation
metabolites of AE, and the mass spectra revealed the indicative characteristic product
ions related to the loss of GSH portion (Figures 4D and 5D). Primary fragment ion
m/z 317 is a result that m/z 592 lost the GSH portion but without the loss of the sulfur
atom (−273 Da). The cleavage of the glycinyl portion (−75 Da) and glutaminyl
section (−146 Da) from m/z 592 resulted in the formation of fragment ions m/z 517
and 446, respectively. Ion m/z 343 consequently came from the deprivation of the
N-formylglycinyl group (103 Da) derived from product ion m/z 446. The observed
fragment ion m/z 463 was a result of the neutral loss (NL) of the γ-glutamyl portion
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(129 Da) from fragment m/z 592.
A trace amount of M6 was observed in
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microsomal reaction without incorporation of NADPH (Figure 5A), perhaps resulting
from the product of AE auto-oxidation. Additionally, the [M+H]+ of M5 and M6
(m/z 592) was 16 Da more than that of M3 and M4, and the product ions of M3 (m/z
301) and M4 (m/z 501) were both 16 Da less than that of M5 (m/z 317) and M6 (m/z
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517), respectively.
This suggests that M5 and M6 were generated from the
conjugation of hydroxylation metabolites of AE with a molecule of GSH.
To identify the metabolic pathways of M5 and M6 (GSH conjugates), synthetic
M1 and M2 were directly and individually mixed and stirred with GSH.
The
product in the M1-GSH mixture displayed identical chromatographic behavior and
fragment pattern as that of M5 observed in microsomal reactions (Figure 4C and E).
On the other hand, the product obtained in the M2-GSH mixture and M6 also revealed
identical retention time and mass spectrometric identities (Figure 5C and E). This
allows us to propose that M5 and M6 resulted from the conjugation of a molecule of
GSH with M1 and M2, respectively. Unfortunately, we failed to obtain pure M5 and
M6 for NMR characterization, since the two GSH conjugates were decomposed
during the process of purification.
Formation of Oxidative Metabolites in Primary Hepatocytes.
To investigate the metabolism of AE in hepatocytes, oxidative metabolites
generated in cultured primary rat hepatocytes were characterized using LC–MS/MS.
Two oxidative metabolites were found in primary hepatocytes exposed to AE (Figure
6B). As expected, such metabolites were not found in primary hepatocytes treated
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with vehicle (Figure 6A).
Similar observation in retention time and fragment
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patterns of the oxidative metabolites in hepatocytes was obtained with those of M1
and M2 generated in both chemical synthesis and microsomal reactions (Figures 2E, F
and 6C, D).
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Four metabolites (tentatively M3/M4 and M5/M6) displaying their [M+H]+ at
m/z 576 and 592 were observed in cultured rat hepatocytes after exposure to AE
(Figures 7B and 8B). The four metabolites are considered to be GSH conjugates
originating from AE and hydroxylated AE, respectively, based on the [M+H]+ of m/z
576 and 592 observed. In addition, the metabolites found in the cells demonstrated
the same chromatographic identity and fragment patterns as those of M3/M4 and
M5/M6 observed in microsomal reactions (Figures 7C, D and 8C, D). As expected,
none of the GSH conjugates was observed in hepatocytes treated with vehicle
(Figures 7A and 8A).
Biliary and Urinary Metabolites of AE
In vivo AE metabolism study was carried out in rats. Bile and urine samples
were collected, followed by LC-MS/MS analysis. No AE-derived GSH conjugates
were observed in blank bile samples (Figure 9A, B), while three GSH conjugates (M3,
M4 and M6) with [M+H]+ at m/z 576 and 327 (M3 and M4), m/z 592 and 317 (M6)
were found in the bile of the animals treated with AE (Figure 9C, D).
The
metabolites observed in vivo study (Figure 9C, D, E, F and G) were consistent with
those found in microsomal incubations (Figures 3B, D, E, and 5B, D) in aspects of
chromatographic and mass spectrometric behaviors. However, we failed to find M5
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(a hydroxylated AE GSH conjugate) from the bile of rats treated with AE.
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Hydroxylation metabolites M1 and M2 were observed in urine samples
obtained from rats after an intraperitoneal injection of AE (Figure 10B, C), and
neither of the two metabolites was found in blank urine (Figure 10A). The urinary
metabolites demonstrated similarity in their retention time and major fragmentation
patterns (Figure 10B, C, D and E) as that of the corresponding metabolites produced
in microsomal reactions (Figure 2B, E and F). Nevertheless, the analysis of urine
samples from AE treated rats showed no AE-derived GSH conjugates. Instead, a
new metabolite (M7) was detected with [M+H]+ at m/z 448 in agreement with the
molecular weight of the corresponding NAC conjugate, i.e. the conjugate composed
of a molecule of hydroxylation metabolite of AE and a molecule of NAC (Figure
11B). Again, the conjugate was not found in urine of rats given vehicle (Figure
11A).
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As shown in Figure 11B, urinary M7 eluted at 9.63 min and was detected by
scanning precursor/product ion pairs m/z 448 and 317 in positive ion mode. Primary
fragment ion m/z 317 was derived from the loss of NAC without sulfur from m/z 448.
Fragment ion m/z 343 was generated from the loss of acetylamino and carboxylic acid
portions (105 Da) from m/z 448. Product ion m/z 130 originated from NAC moiety
absent of sulfur. Product ion m/z 319 resulted from the neutral loss of the glutamyl
moiety (−129 Da) from m/z 448. This suggests that M7 was the conjugate composed
of a molecule of NAC and a molecule of oxidative metabolite of AE. As expected,
an NAC conjugate was observed in an AE- and NADPH-fortified rat microsomal
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incubations trapped with NAC. The NAC conjugate was found to share the identical
chromatographic and mass spectrometric properties with the urinary NAC conjugate,
as shown in Figure 11C, E and D.
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P450 Enzymes Participating in AE Metabolism.
Characterization of P450 enzymes participating in metabolic AE hydroxylation
was achieved by monitoring the production of M1, M2, M5 and M6 in incubation of
AE with individual recombinant human P450 enzymes fortified with NADPH and
GSH. P450s 1A2, 3A4, and 3A5 displayed the catalytic property in the production
of M1, M2 and M5.
Additionally, P450 1A2 was found to show the most
effectiveness for the catalytic hydroxylation among the three (Figure 12A, B).
However, the generation of M6 can apparently be “catalyzed” by all P450 enzymes
tested. We found that M6 was derived from M2 some of which possibly came from
auto-oxidation of AE. In other word, partial M6 might result from the spontaneous
reaction of GSH with contaminated M2.
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DISCUSSION
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We attempted to study in vitro biotransformation of AE to better understand the
biochemical mechanism of AE cytotoxicity. Initially, the fragmentation pattern of
parent compound AE were explored (Figure 1), which assists us to characterize the
structure of AE metabolites.
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Two hydroxylation metabolites (M1 and M2) were observed in incubation
mixture containing AE and rat or human liver microsomes. The amount of M1
produced in human liver microsomal (HLM) incubation system was about three times
higher than that in the rat liver microsomal (RLM) system. The amount of M2
produced in HLMs was about two times higher than that produced in RLMs (Figures
S1-3). Apparently, human liver microsomes showed higher activity then rat liver
microsomes to catalyze the hydroxylation of AE. NADPH was required for the
formation of the two oxidative metabolites. As shown in Scheme 1, the oxidative
metabolism and GSH conjugation pathways of AE is proposed from the obtained
mass spectrometric data. M1 and M2 were chemically synthesized to verify the
structures of the oxidative metabolites. M2 was assigned as 5-hydroxy aloe-emodin
based on its NMR data (Figure S4). Unfortunately, we failed to gain a quality of
NMR spectrum of M1 for structural identification, which might result from the
instability of M1. High resolution mass spectrum of M1 demonstrated its protonated
molecule ion of m/z 287.0549 (Table 1), which agrees with the molecular weight of
monohydroxylation of AE.
The assignment of M2 as 5-hydroxy aloe-emodin
enables us to propose that M1 was 7-hydroxy aloe-emodin, since hydroxyl substitutes
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with electron donation property are well known to activate ortho- and para-carbons
for electrophilic aromatic substitution.
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Four GSH conjugates (M3-M6) were observed in NADPH- and GSH-fortified
rat and human liver microsomes. M3 and M4 were found in the reaction system
without NADPH (Figure 3A), demonstrating that P450 enzymes did not participate in
the generation of M3 and M4. We anticipate that M3 and M4 were generated from
direct reaction of AE with GSH. To verify the speculation, we allowed AE to react
GSH in a buffer system. As expected, two products derived from GSH showed
identical retention time and mass spectral identity (Figure 3C, F and G) as those of
M3 and M4 found in microsomal reactions. M3 and M4 are most likely positional
isomers resulting from adduction of GSH on different carbons of the aromatic ring of
AE. Together, the results indicate that AE reacted with nucleophilic GSH without
the assistance of enzyme catalysis. This enables us to speculate that AE can form
protein covalent binding via the direct reaction of AE with cysteine residues of
protein. The [M]+ of M5 and M6 (m/z 592) were 16 Da higher than that of M3 and
M4 (m/z 576), suggesting that M5 and M6 resulted from oxidative metabolites of AE.
This encouraged us to define the metabolic routes of the two GSH conjugates by
mixing of GSH with M1 or M2. We found that M5 originated from M1, and M6
came from M2 (Figures 4 C, E and 5 C, E).
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Additionally, AE metabolism study was carried out in primary rat hepatocytes.
Six metabolites, including M1−M6 as observed in microsomal incubations, were
detected in primary rat hepatocytes treated with AE. GSH conjugates resulting from
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direct adduction of AE or its hydroxylation metabolites with GSH were detected in
AE-exposed cells, further providing the evidence for the reactivity of AE and
oxidative metabolites of AE toward sulfhydryl-containing nucleophiles. The GSH
conjugates observed in microsomal incubation and cultured primary hepatic cell
exposure studies imply that AE and AE hydroxylation metabolites covalently bind
to cysteine residues of proteins or other thiol-containing biomolecules, which could
initiate the toxicities of AE and AE oxidative metabolites.
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Two AE GSH conjugates (M3 and M4) and one hydroxylated AE GSH
conjugate (M6) were observed in bile samples of animals treated with AE as detected
in rat liver microsomal reactions. Unexpectedly, M5 was not detected in the bile.
Inconsistence of in vitro vs in vivo data often takes place in drug metabolism studies,
particularly that in microsomal vs animal studies. Microsomes contain limited drug
metabolizing enzymes compared with that of animal models. In the present study,
the microsomal incubations were set for P450-mediated oxidation reactions. GSH
conjugates M3-M6 could further be metabolized by enzymes which were either not
included in microsomes or were unable to work under the particular condition. This
may explain the disagreement observed in the in vitro vs in vivo studies. GSH
conjugates excreted in bile are generally employed as biomarkers of exposure to
electrophilic agents.24, 25 The observed GSH conjugates indicate that AE and its
hydroxylation metabolite are all electrophilic species that have chemical reactivity
toward the sulfhydryl of GSH to generate the corresponding GSH conjugates. It is
most likely that these electrophiles alkylate cysteine-based protein. Protein covalent
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binding has been considered as one of biochemical mechanisms by which various
drugs and chemicals induce toxicities.26, 27 This made us propose that modification
of protein critical for cell maintenance may trigger the development of the
cytotoxicity of AE. However, profound research needs to be done to verify the
hypothesis. In addition, GSH conjugates are commonly metabolized to mercapturic
acids which are then excreted in urine. It is reasonable to find NAC conjugate M7 in
urine samples of rats given AE.
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Given together, we propose the oxidative metabolism and GSH conjugation
pathways of AE as shown in Scheme 1. The 1, 4-quinone moiety makes the parent
compound reactive to thiols. AE directly reacts with GSH through Michael addition
to generate GSH conjugate 1. The conjugate is sequentially oxidized to M3/M4 via
auto-oxidation. Additionally, AE undergoes P450-mediated hydroxylation to form
M1 and M2, followed by GSH conjugation. The resulting GSH conjugates (2 and 3)
are further oxidized to M5 and M6 as for the formation of M3/M4.
In conclusion, metabolic oxidation of AE produced two phase I metabolites,
including 5-hydroxy aloe-emodin and possibly 7-hydroxy aloe-emodin.
P450s 1A2,
3A4 and 3A5 participated in the observed AE hydroxylation. Both AE itself and
hydroxylation metabolites are electrophilic species that have chemical reactivity to
GSH.
The findings certainly assist the understanding of the mechanisms of
AE-induced cytotoxicity.
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FUNDING SOURCES
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This work was partly supported by the National Natural Science Foundation of China
[Grant 81430086, 81773813, and 81830104].
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SUPPORTING INFORMATION
LC-MS/MS analysis of M1-M6 in rat liver microsomal incubations; Tandem spectra
of M1-M6 produced in rat microsomal reactions; 1H NMR spectrum of M2.
ABBREVIATIONS
CE, collision energy; DMSO, dimethyl sulfoxide; DP, declustering potential; EPI,
enhanced product ion; GSH, glutathione; IDA, information-dependent acquisition;
LC-MS/MS, liquid chromatography-tandem mass spectrometry; MRM,
multiple-reaction monitoring; NAC, N-acetylcysteine; NADPH, β -nicotinamide
adenine dinucleotide 2’-phosphate reduced tetrasodium salt; FBS, fetal calf serum;
DMEM, dulbecco's modified eagle's medium; HLMs, human liver microsomes;
RLMs, rat liver microsomes; NMR, nuclear magnetic resonance.
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Table 1. Mass spectrometric data acquired from high resolution mass spectrometric
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analysis of synthetic M1.
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Figure Legends
Figure 1. Tandem mass spectrum of AE.
Figure 2. Characterization of M1 and M2. Ion chromatograms (m/z 287 → 241)
gained from LC-MS/MS analysis of AE-fortified human liver microsomal reactions in
the absence (A) or presence (B) of NADPH. Ion chromatograms (m/z 287→241)
gained from LC-MS/MS analysis of synthetic M1 (C) and M2 (D). Tandem mass
spectra of M1 (E) and M2 (F) produced in human liver microsomal reactions.
Tandem mass spectra of synthetic M1 (G) and M2 (H).
Figure 3. Characterization of M3 and M4. Ion chromatograms (M3, m/z 576→301;
M4, m/z 576 → 327) gained from LC-MS/MS analysis of AE-fortified human liver
microsomal reactions supplemented with GSH in the absence (A) or presence (B) of
NADPH.
C: Ion chromatogram (m/z 576 → 301/327) gained from LC-MS/MS
D: Tandem spectrum of M3 generated in
analysis of synthetic M3 and M4.
microsomal reactions. E: Tandem mass spectrum of M4 generated in microsomal
reactions. F: Tandem mass spectrum of synthetic M3. G: Tandem mass spectrum
of synthetic M4.
Figure 4. Characterization of M5. Ion chromatogram (m/z 592→317 ) gained from
LC-MS/MS analysis of AE-fortified human liver microsomal reactions supplemented
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