Metabolic Activation of Rhein: Insights into the Potential Toxicity Induced by Rhein-Containing Herbs

Article abstract of DOI:10.1021/acs.jafc.6b01872

Rhein is a major component of the many medicinal herbs such as rhubarb. Despite wide use, intoxication cases associated with rhein-containing herbs are often reported. The present work aimed to investigate if rhein was subject to metabolic activation leading to toxicity. Upon incubations with different species of liver microsomes, three monoglucuronides were identified, corresponding to two hydroxyl glucuronides and one acyl glucuronide via the carboxyl group, respectively. Further study revealed that rhein acyl glucuronide was chemically reactive, and showed cytotoxicity toward hepatocarcinoma cells. In addition, significant species differences in glucuronidation of rhein were observed between laboratory animals and humans. Reaction phenotyping experiments demonstrated that rhein acyl glucuronide was catalyzed predominantly by uridine 5′-diphospho-glucuronosyltransferase 1A1, 1A9, and 2B7. Taken together, the present study confirmed that rhein could be metabolically activated via the formation of acyl glucuronide, especially in human.

Full text of DOI:10.1021/acs.jafc.6b01872

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Article
Metabolic activation of rhein: insights into the
potential toxicity induced by rhein-containing herbs
Yuan Yuan, Jiyue Zheng, Meiyu Wang, Yuan Li, Jianqing Ruan, and Hongjian Zhang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01872 • Publication Date (Web): 30 Jun 2016
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Page 1 of 34
Journal of Agricultural and Food Chemistry
Metabolic activation of rhein: insights into the potential toxicity
induced by rhein-containing herbs
Yuan Yuan1, Jiyue Zheng, Meiyu Wang, Yuan Li, Jianqing Ruan*, Hongjian Zhang*
1College of Pharmaceutical Sciences, Soochow University, Suzhou, China
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Abstract
Rhein is a major component of the many medicinal herbs such as rhubarb.
Despite of wide use, intoxication cases associated with rheinꢀcontaining herbs are
often reported. The present work aimed to investigate if rhein was subject to
metabolic activation leading to toxicity. Upon incubations with different species of
liver microsomes, three monoꢀglucuronides were identified, corresponding to two
hydroxylꢀglucuronides and one acyl glucuronide via the carboxyl group, respectively.
Further study revealed that rhein acyl glucuronide was chemically reactive, and
showed cytotoxicity towards hepatocarcinoma cells. In addition, significant species
differences in glucuronidation of rhein were observed between laboratory animals and
humans. Reaction phenotyping experiments demonstrated that rhein acyl glucuronide
was catalyzed predominantly by Uridine 5'ꢀdiphosphoꢀglucuronosyltransferase 1A1,
1A9 and 2B7. Taken together, the present study confirmed that rhein could be
metabolically activated via the formation of acyl glucuronide, especially in human.
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Key words: rhein, metabolic activation, acyl glucuronide
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INTRODUCTION
Anthraquinone compound rhein, is widely distributed in many medicinal and
nutritional plants including Rheum palmatum, Polygonum multiflorum, Cassia
angustifolia and Cassia occidentalis (1). These rheinꢀcontaining herbs were widely
used for the antiꢀinflammatory, antidotal, antipyretic and laxative properties in Asia
for centuries in countries of China, Japan, and Korea (2-4). In spite of wide use, the
relevant intoxication cases due to the consumption of these herbs were reported
continuously (5). Recently, the association between children death and consumption
of Cassia occidentalis seeds has been reported (6). The anthraquinones were proved
to be the major chemicals in the Cassia occidentalis seeds responsible for producing
toxicity and the most cytotoxic moiety in rat primary hepatocytes and
hepatocarcinoma cell line (HepG2) among all the anthraquinones was rhein (7).
Polygonum multiflorum preparation, which also contained rhein and other
anthraquinones, was proved to cause acute liver injury as documented in LiverTox
website organized by the National Institutes of Health (8).
The toxicity of rhein has been reported to be associated with redox cycling and
nucleophilic addition reactions with biomolecules (9-11). Rhein was demonstrated to
be able to interfere with a number of mitochondrial functions, such as inhibition of the
oxidation of nicotinamide adenine dinucleotide ꢀ or flavin adenine dinucleotide
ꢀlinked substrates and oxidative phosphorylation (12-14). It has been reported that
rhein was involved in the nucleophilic addition of thiols leading to a depletion of
reduced glutathione (15). In addition, rhein has been found to alter the cytoskeleton by
affecting the plasma membrane and intracellular membranes (16, 17).
Rhein underwent extensive metabolism in the liver, in particular glucuronidation
(18, 19) and thus exhibited low oral bioavailability in rats and humans (20). The
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major circulating form in the plasma after oral administration of rheinꢀcontaining
products should be the corresponding glucuronides rather than the parent compound.
However, the toxicity studies were all focused on the parent rhein (21, 22). As a
carboxylic anthraquinone, rhein has the potential to be metabolized into rhein acyl
glucuronide. It was reported that acyl glucuronides could covalently modify
endogenous proteins due to their electrophilic capacity that cause substitution
reactions with the nucleophilic groups located on proteins or other macromolecules,
and this reaction could ultimately lead to adverse drug toxicities associated with
carboxylic acidꢀcontaining drugs (23).
Most of the toxicity studies of rhein have been performed using parent
compound against rat’s primary hepatocytes or other cell lines (21-24). However, the
toxicity was caused by the parent compound directly or by the metabolites remains
unknown. In addition, although a number of studies demonstrated that rhein
underwent extensive glucuronidation in the liver (25), the formation of rhein acyl
glucuronide and the reactivity of rhein acyl glucuronide were still unknown.
Therefore, this work was designed to study the metabolic activation of rhein
through acyl glucuronide formation and the cytotoxicity of rhein acyl glucuronide. In
addition, the regioselectivity of rhein glucuronidation by different human Uridine
5'ꢀdiphosphoꢀglucuronosyltransferases (UGTs) was also conducted to find the major
UGTs responsible for the metabolic activation of rhein.
MATERIALS AND METHODS
Materials
Rhein and hyperoside (internal standard, IS) were obtained from Shanghai
Jiukun International Trade Co., Ltd. (Shanghai, China). Alamethicin, LꢀGlutathione
reduced, DꢀSaccharic acid 1, 4ꢀlactone monohydrate and formic acid were obtained
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from Sigma (St. Louis, MO). Uridine diphosphate glucuronic acid (UDPGA) was
purchased from Roche (Basel, Switzerland). Dimethyl sulfoxide, disodium hydrogen
phosphate and sodium dihydrogen phosphate were purchased from Sinopharm
Chemical Reagent co., Ltd. (Shanghai, China). Acetonitrile of HPLC (high
performance liquid chromatography) grade were purchased from Merck (Darmstadt,
Germany). Ultraꢀwater was purified by Hitech Laboratory water purification systems
(Shanghai, China).
Human liver microsomes (HLMs, pooled from 20 different organ donors), mouse,
dog and monkey liver microsomes and recombinant human UGT isoforms (UGT1A1,
UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4,
UGT2B7, UGT2B15, and UGT2B17) expressed in baculovirusꢀinfected insect cells
were purchased from BD Gentest (Woburn, MA). Pooled SpragueꢀDawley rat liver
microsomes were prepared using standard methods as described previously (26). The
research protocol was approved by the animal care and use committee at Soochow
University.
Synthesis and NMR analysis of Rhein Acyl Glucuronide
The synthesis of rhein acyl glucuronide was outlined in Figure 1. Condensation
of rhein 1 with allyl glucuronate 2 (27) using the procedure developed by Bowkett (28)
1
gave the desired conjugate 3 in 30% yield. The H NMR signal of the anomeric
proton (δ 5.8, 1H) indicates that compound 3 is the desired βꢀanomer product (28). To
remove the allyl protection, compound 3 was treated with Pd(PPh3)4 and morpholine
as reported (28). The deprotect product was precipitated from the solvent during the
reaction time as a mophine salt, which was filtered and acidified with Amberlyst Aꢀ15
(H+) to give the free acyl glucuronide 4. Reaction progress was monitored by
analytical thin layer chromatography performed on silica gel preꢀcoated plates.
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Column chromatography was prefromed with silica gel (100ꢀ200 mesh, Qingdao
Marine Chemical Inc.,Qingdao,China).. 1H NMR and 13C NMR spectra were obtained
on 400 MHz (Varian) spectrometers. Chemical shifts were given in ppm using
tetramethylsilane (TMS) as internal standard. Mass spectra were obtained using an
Agilent 1100 LC/MSD Trap SL version mass spectrometer.
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To a solution of rhein (568 mg, 2.0 mmol) and allyl glucuronate 2 (468 mg, 2.0
mmol) in MeCN (20 mL) and THF (10 mL) was added HATU (760 mg, 2.0 mmol)
and Nꢀmethylmorpholine (404 mg, 4.0 mmol). The mixture was stirred at room
temperature under nitrogen for 8 h, and then quenched by addition of Amberlyst Aꢀ15
(H+, 2 equiv). After evaporation the resulting residue was chromatographed, eluting
with 10% Ethanolꢀ dichloromethane to give the title compound 3 as a yellow solid
(300 mg, 30%). 1H NMR (400 MHz, CDCl3) δ 8.16 (s, 1H), 7.90 (s, 1H), 7.82 (d, J =
7.2 Hz, 1H), 7.74 (d, J = 6.8 Hz, 1H), 7.41 (d, J = 8.0 Hz, 1H), 5.96ꢀ5.85 (m, 1H),
5.74 (d, J = 7.2 Hz, 2H), 5.55 (d, J = 5.2 Hz, 1H), 5.41 (br s, 1H), 5.34 (d, J = 17.6 Hz,
1H), 5.22 (d, J = 10.4 Hz, 1H), 4.62 (d, J = 4.8 Hz, 2H), 4.10 (d, J = 8.8 Hz, 1H),
3.53ꢀ3.42 (m, 3H).
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To a solution of 3 (200 mg, 0.4 mmol) and Pd(PPh3)4 (46 mg, 0.04 mmol) in
tetrahydrofuran (5 mL) under nitrogen at 0 °C was added morpholine (72 mg, 0.8
mmol). The reaction mixture was stirred at the same temperature for 2 h. The
morpholine salt of compound 4 was precipitated from the solvent during the reaction
time. After filtered and washed with diethyl ether (3 mL x 2), the yellow solid was
suspended in tetrahydrofuran (5 mL) and Amberlyst Aꢀ15 (H+, 50 mg) was added.
After stirring samples at room temperature for 1 h, the reaction mixture was filtered
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and concentrated to afford the title compound 4 as a yellow solid (65 mg, 35%). H
NMR (400 MHz, DMSOꢀd6) δ 11.89 (br s, 2H), 8.19 (s, 1H), 7.92 (s, 1H), 7.84 (dd, J
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= 8.0, 8.0 Hz, 1H), 7.46 (d, J = 7.6 Hz, 1H), 7.43 (d, J = 7.6 Hz, 1H), 5.71ꢀ5.63 (m,
2H), 5.35 (br s, 1H), 3.87 (d, J = 8.8 Hz, 1H), 3.46ꢀ3.38 (m, 3H).
Glucuronidation of Rhein in Liver Microsomes
Rhein at 25 ꢁM was incubated with 5 mM MgCl2, 20 ꢁg/mL alamethicin, 5 mM
Dꢀsaccharicacid 1,4ꢀlactonein 50 mM phosphate buffer (pH 7.4) in the presence of 1
mg/mL human liver microsomes, mouse liver microsomes, monkey liver microsomes,
dog liver microsomes or 1.64 mg/mL rat liver microsomes. The reaction was initiated
by adding 2 mM UDPGA and kept at 37 °C for 40 min.
Stability of different Rhein Glucuronides in incubation and sample solution
Rhein at 100 ꢁM was incubated with 1 mg/mL HLMs for 40 min at the same
condition described above. The incubation solution was prepared by direct
centrifugation of the above incubation mixture at 13,000 g for 10 min, while sample
solution was prepared by adding 3 volume of iceꢀcold methanol containing 3% formic
acid before further centrifugation. The corresponding supernatant was transferred into
new tubes to test the rhein glucuronides stability in incubation and sample solution,
respectively. Aliquots (40 ꢁL) of the supernatants were further kept at 37 °C for
different time intervals. At the time point of 0, 2, 4, 6, 8 and 24 h, 120 ꢁL iceꢀcold
methanol containing 3% formic acid and IS was added into the incubation solution to
quench the degradation.
In vitro Reactivity of Rhein Acyl Glucuronide with glutathione
15 ꢁM of RG3 was dissolved in phosphate buffered saline (PBS, 50 mM, pH 7.4)
in the presence or absence of glutathione (GSH, 10 mM) and kept at 37 °C for
different time intervals according to the reference (29). Aliquots of 150 ꢁL incubation
mixture were taken and added to three volume of iceꢀcold methanol containing 3%
formic acid to quench the reaction.
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Glucuronidation Activity of Recombinant Human UGTs towards Rhein
Glucuronidation activity of rhein in pooled HLMs and 12 recombinant human
UGTs (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15 and 2B17)
was measured in the aforementioned glucuronidation reaction system which contained
0.1 mg/mL pooled HLMs or individual recombinant human UGTs instead. All
reactions were conducted in triplicate at 37 °C for 40 min.
Kinetics of Rhein Glucuronidation in Pooled HLMs and Recombinant Human
UGTs
Kinetics of rhein glucuronidation was determined under the initial rate conditions.
Briefly, rhein (0.2ꢀ100 ꢁM) was incubated with 0.1 mg/mL of pooled HLMs or
selected individual UGT Supersomes (UGT1A1, 1A7, 1A8, 1A9 and 2B7) in the
presence of 2 mM UDPGA in a total volume of 0.1 mL at 37 °C for 40 min. Each
experiment was conducted in triplicate.
Sample Preparation
All the glucuronidation reactions were terminated by adding 3 volume of
iceꢀcold methanol containing 3% formic acid and the IS. The mixtures were vortexed
immediately followed by centrifugation at 13,000 g for 10 min. Aliquots of the
supernatants were subjected to LCꢀMS/MS. The metabolites and rhein were all
showed desirable stability in the acidic methanol solution at least for 40 h.
Cytotoxicity of rhein and rhein acyl glucuronide
HepG2 cells were obtained from American type culture collection. Cells were
grown in dulbecco's modified eagle medium supplemented with 10% fetal bovine
serum and 1% penicillin ꢀstreptomycin solution in a humidified atmosphere of 5%
CO2/95% air at 37 °C. 1 × 104 cells/well (in 120 ꢁL of medium) was transferred to a
96ꢀwell plate. Cells were allowed to attach for 24 h and treated with different
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concentrations of rhein and RG3. After 24 h of treatment, 100 ꢁg of 3ꢀ
（4,5ꢀdimethylꢀ2ꢀthiazolyl)ꢀ2,5ꢀdiphenylꢀ2ꢀHꢀtetrazolium bromide (MTT) was added
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into each well and incubations continued for 4 h. In the end, the medium was removed
and the MTT formazan was further dissolved in dimethylsulfoxide. The absorbance
was determined at 490 nm by a microplate reader (BioTek, Winooski, VT).
LC-MS/MS Analysis
All samples were analyzed by a LCꢀMS/MS system consisting of an API4000
Qtrap mass spectrometer equipped with a turboꢀV ionization source (Applied
Biosystems, Foster City, CA, USA), two LCꢀ20AD pumps with a CBMꢀ20A
controller, DGUꢀ20A solvent degasser and a SILꢀ20A autosampler (Shimadzu,
Columbia, MD, USA). The mobile phase consisted of 0.1% formic acid water (A) and
acetonitrile (B). Samples were analyzed on an AgelaVenusil XBP C18 column (50×2.1
mm, 3 ꢁm) adopting a gradient elution as follows: 0ꢀ0.5 min, 10% B; 0.5ꢀ1.5 min,
10ꢀ30% B; 1.5ꢀ6.0 min, 30ꢀ70% B; 6.0ꢀ7.0 min 70ꢀ100% B; 7.0ꢀ8.0 min 100ꢀ10% B;
8ꢀ10 min, 10% B. Negative ionization mode was chosen for sample analysis. The
MS/MS parameters were set as follows: curtain gas, 30 psi; nebulizer gas (GS1), 55
psi; turbo gas (GS2), 55 psi; ion spray voltage, 4500 V; and ion source temperature,
450 °C. In the selected ion transitions, collision energy and declustering potential
values were set as ꢀ40 and ꢀ24 V for rhein; ꢀ38 and ꢀ14 V for rhein glucuronides; ꢀ81
and ꢀ38 for hyperoside (IS), respectively. Ion transitions were monitored as follows:
rhein, 283.0→239.5; rhein glucuronides, 459.0→283.0; hyperoside, 463.1→300.0.
For the method validation, intraday (three replicates within a day) and interday
(three replicates per day over 3 days) variations were measured at two concentration
levels to determine the precision and accuracy. Both RG3 and rhein showed good
stability in the solution of methanol containing 3% formic acid (90% left after 72 hr
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stock). The calibration curves were constructed by plotting the peak area ratio of the
analyte to the IS. A good linearity (r2 = 0.999) in the concentration range of
100ꢀ25000 nM and 10ꢀ5000 nM was obtained for rhein and RG3, respectively. At the
concentrations tested (rhein: 500, 50000 nM; RG3: 50, 500 nM), intraꢀ and interꢀday
variations were 1%ꢀ11 % and 2%ꢀ5% for rhein and RG3 respectively, demonstrating
an adequate reproducibility in the concentration range analyzed. In addition, great
accuracies of 85.3%ꢀ105.0% were achieved at these these concentrations examined.
Data Analysis
All data were presented as means ± standard deviation (S.D.). Apparent kinetics
by UGT Supersomes was obtained by fitting the experimental data with
MichaelisꢀMenten (Eq. (1)) model with nonlinear regression analysis using Prism 5.0
(GraphPad Software, CA). Assuming a wellꢀstirred model, the in vitro intrinsic
hepatic clearance (CLint) was then calculated with the Eq. (2).
V = (Vmax× [S])/ (Km + [S]) (1)
CLint = Vmax/ Km (2)
Where v is the velocity of metabolite formation, Vmax is the maximum velocity, Km is
the Michaelis constant defined as the substrate concentration at half of Vmax, [S] is the
substrate concentration and CLint is the intrinsic clearance of liver.
RESULTS
Glucuronidation of Rhein in Different Liver Microsomes
When rhein was incubated with rat, mouse, dog, monkey or human liver
microsomes in the presence of UDPGA, three additional peaks with the ion transition
of 459.0→283.0 were observed in all incubations (Figure 2A & 2B),which were
absent in controls. These three peaks (named as RG1, RG2 and RG3) all showed the
molecular ion at m/z 459, 176 mass units higher than that of rhein (m/z 283) (Figure
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2C). In addition, the MS2 spectrum showed characteristic [MꢀGlcAꢀH]ꢀ at m/z 283,
corresponding to loss of one molecular of glucuronic acid. As a result, these
metabolites were tentatively identified as three monoꢀglucuronides of rhein,
corresponding to two monoꢀglucuronides of two hydroxyl groups and one
carboxyl group, respectively. Among these three peaks, RG3 showed the same
retention time and mass spectra as those of synthesized rhein acyl glucuronide.
Therefore, the structure of RG3 was unambiguously identified as rhein acyl
glucuronide. In contrast, RG1 and RG2 were tentatively assigned as rhein hydroxyl
glucuronides.
The formation rates of three monoꢀglucuronides varied significantly among
different species. As shown in Figure 2B, Rhein acyl glucuronide (RG3) was found as
the predominant metabolite accounting for about 60% of total glucuronides in both
human and monkey liver microsomes (UV reponse showed similar pattern). However,
in rat, mouse and dog liver microsomes, RG3 was the lowest metabolites among three
glucuronides.
Stability of different Rhein Glucuronides in incubation solution
After incubation of rhein with human liver micosomes for 40 min, the protein
was removed by centrifugation producing the resulting incubation solution. The
stability of three glucuronides of rhein was further investigated for 24 h. As shown in
Figure 3A, in the incubation solution, RG1 and RG2 remained constant within 24h.
However, RG3 decreased along with time and only 54% ± 0.15% remained in the
incubation solution after 24 h.
The stability of RG3 was further evaluated using standard solely in PBS buffer.
As shown in Figure 3B, RG3 standard was not stable in PBS either and it was rapidly
transformed into RG4 in the first two hours. Thereafter, the formed RG4 and
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remaining RG3 further transformed into rhein along with time, indicating the
instability of RG3.
Reactivity of Rhein Acyl Glucuronide with GSH in Vitro
Incubation of synthesized RG3 with GSH in phosphate buffer (pH 7.4) at 37 °C
resulted in rapid decrease of RG3 as shown in Figure 4A&B. Meanwhile, one peak
with ion transition of 459.0→283.0 (RG4) and another one with 445→267 (RGSH) as
well as rhein appeared and increase rapidly after incubation. The peak RG4 showed
the same ion transition as that of RG3, but with different retention time from RG1,
RG2 and RG3, indicating that RG4 might be an isomer of RG3 generated by
intramolecular migration. The peak RGSH produced a quasiꢀmolecular ion at m/z
445.4 at positive model as shown in Figure 4C, 129 Da less than the molecular ion of
rhein GSH conjugate. Neural loss of 129 Da is the characteristic mass pattern of GSH
conjugates at positive model, due to the loss of pyroglutamic acid (29), indicating
RGSH might be a GSH conjugate. Moreover, the MS2 of RGSH showed product ion
+
at m/z 267 (Figure 4C), which was the [M+HꢀGSH] ion of rhein GSH conjugate.
These results demonstrated that RGSH was the monoꢀGSH conjugate of rhein.
Therefore, RG3 was proved to be chemically reactive by binding with GSH.
Identification of UGT Isoforms Involved in Glucuronidation of Rhein
The relative activities of 12 recombinant human UGT isoforms (UGT 1A1, 1A3,
1A4、1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15 and 2B17) expressed in insect
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cells were determined in terms of the formation of rhein glucuronides at 25 ꢁM. The
formation rates of rhein glucuronides after incubation of rhein with UGT isoforms
were shown in Figure 5. UGT1A1, 1A7, 1A8, 1A9 and 1A10 catalyzed RG1
formation, and UGT1A1, 1A7, 1A8 and 1A9 showed activity towards RG2 formation.
RG3 formation was mediated predominantly by UGT 1A1, 1A9 and 2B7. UGT1A1
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showed the highest activity, followed by 1A9 and 2B7, towards RG3 formation. UGT
1A8 is the most potent isform mediating RG1 formation followed by UGT 1A1, 1A7
and 1A9, while UGT 1A8 showed the highest activity towards RG2 followed by UGT
1A9, 1A7 and 1A1. More interestingly, UGT 2B7 showed preference of
glucuronidation at the carboxyl group.
Kinetics of rhein glucuronidation in HLMs and recombitant UGTs
The effects of the concentration of rhein on its glucuronidation activity by HLMs
and five individual human UGTs were investigated as shown in Figure 6. The Vmax
and Km as well as the Clint, which were calculated from the formations of RG1, RG2
or RG3, are summarized in Table 1.
The RG3 was the major metabolite of rhein formed in HLMs within the tested
substrate concentration range (0.2ꢀ100 ꢁM), indicating that HLMs preferentially
catalyze glucuronidation of rhein at the carboxyl group, rather than the hydroxyl
group. All of RG1, RG2 and RG3 formations by HLMs followed typical
MichaelisꢀMenten kinetics. HLMs exhibited the highest Vmax for the formation of
RG3 among the three glucuronides, but similar Km for the formation of RG1, RG2
and RG3 (77.4, 52.2 and 50.0 for RG1, RG2 and RG3, respectively). As such, the
apparent intrinsic clearance of rhein in HLMs through RG3 was much higher than
RG1 and RG2.
Within the substrate concentration range tested, only UGT 1A1, 1A9 and 2B7
catalyzed RG3 formation from rhein. The carboxyl glucuronidation by UGT 1A1,
1A9 and 2B7 followed typical MichaelisꢀMenten kinetics. UGT 1A1 displayed the
highest capacity in converting rhein to RG3 with the lowest Km (14.81 ꢁM) and the
highest Vmax, followed by 1A9 (Km 20.86 ꢁM) and 2B7 (Km 66.77 ꢁM).
UGT 1A1, 1A7, 1A8 and 1A9 catalyzed both RG1 and RG2 formation from
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rhein. The phenolic glucuronation by UGT 1A1, 1A7, 1A8 and 1A9 followed typical
MichaelisꢀMenten kinetics. UGT 1A8 exhibited a highest Vmax for the formation of
RG1 and RG2 as shown in Figure 6.
297
298
Cytotoxicity of rhein acyl glucuronide
As showed in Figure 7, obvious cytotoxicity of rhein and RG3 towards HepG2
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cells was observed at 200 ꢁM. The cytotoxicity effect of RG3 was relatively less than
rhein, demonstrating the less direct toxicity of RG3 to HepG2 cells. 200 ꢁM of RG3
significantly reduced the viability of HepG2 cells to 30.3%, which might be result
from chemical reactivity of RG3 by directly binding with biomacromolecule.
DISSCUSION
The intoxication cases due to consumption of rheinꢀcontaining products in
human beings were reported from time to time (30). More seriously, recent studies
showed that some cases of children death might be associated with the consumption
of Cassia occidentalis seeds and the toxic moieties were proved to be rhein with the
highest cytotoxicity followed by emodin, aloeꢀemodin and other rhubarb
anthraquinones (6, 7, 31). It was reported that oral administration of rhubarb might
lead to hepatotoxicity and nephrotoxicity in rats (32, 33), and mice (34). However,
previous acute and subacute toxicity test, mutagenesis test showed that rhubarb was
safe and nontoxic in rats (35). One recent study also found that oral administration of
4000 mg/kg/d rhubard extracts for 90 days did not cause subchronic toxicity in
Sprague Dawley rats (36). Until now, there is still no agreed conclusion about the
toxicity of rhein in human beings and other species.
The discrepancy in response to oral administration of rhubarb between rats and
human beings might come from the species difference in the metabolic activation of
rhein. Our results showed that rhein was mainly metabolized into the acyl glucuronide
in liver microsomes from primates (human and monkey) which was chemically
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reactive. In contrast, the major metabolites of rhein in liver microsomes from rat,
mouse and dog were monoꢀglucuronides at the hydroxyl groups with no reactivity.
Species differences in regioselectivity of glucuronidation of rhein should be attributed
to varied hepatic UGTs contents across species (37, 38). Due to the species difference
in the metabolic activation of rhein, it may difficult to observe comparative toxic
response from laboratory animals as that shown in human beings. As such, the
monkey should be the suitable animal model for further studies of bioactivation and
toxicity of rhein. It is worthy to note that, in addition to the microsomal enzyme
activity, other factors, such as organ blood flow, total organ mass, also differ between
species and contribute to species difference in vivo.
Rhein underwent extensive phase two metabolism in the liver, especially
glucuronidation. It was reported that, the majority of rhein is eliminated in urine as
glucuronide conjugates (60%), followed by unchanged form (20%) and sulfate
conjugates (20%) after oral administration of rhubarb extract to rats (25). Based on
clinical reports, the Cmax of rhein in patients administered Rheum sp. Ranged from 6.7
to 38.7 mM (39-40), indicating similar or higher exposure level of rhein glucuronides
in human beings. In present study, the major glucuronide generated in HLMs was
proved to be the rhein acyl glucuronide. As a result, the majority of rhein might be
transformed to the acyl glucuronide in the liver, which might be activated and
covalently bind with proteins or DNAs and cause toxicity ultimately.
For the first time, the rhein acyl glucuronide was proved to be chemically
reactive by our study. Rhein acyl glucuronide was not stable in PBS buffer at
physiological temperature. It was transferred to an isomer by intramolecular migration.
More importantly, rhein acyl glucuronide was capable to covalently bind with GSH to
form rheinꢀGSH adduct, indicating its chemical reactivity. Therefore, rhein might
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induce toxicity by metabolic activation to rhein acyl glucuronide and further covalent
binding with endogenous biomacromolecules (42, 43).
For the first time, the circulating metabolite of rhein in the blood (RG3) was
proved to be toxic towards HepG2 cells. Previous cytotoxicity studies all focused on
the direct toxicity of rhein and neglected the potential toxicity of the metabolites,
which might be the main circulating form in the blood. Both direct toxic effects and
immuneꢀmediated toxicity (hypersensitivity reactions) might be the possible
mechanisms of liver injury caused by acyl glucuronides (44). With direct toxicity,
covalent protein binding via acyl glucuronides may disrupt the normal physiological
function of a critical protein and /or DNA or some critical regulatory pathway, leading
to cellular necrosis. Alternatively, the modified protein bound with chemical reactive
acyl glucuronides can act as haptens and initiate an immune reaction that may be
mediated via a specific humoral (antibody) response, a cellular response (T
lymphocytes), or a combination of both (45, 46). Our result revealed that RG3 showed
a relatively lower cytotoxic effect than rhein on HepG2 cell, indicating direct
cytotoxicity might not be the major reason of rhein causing toxicity in human beings.
Although with less direct cytotoxicity than rhein, the activated rhein acyl glucuronides
might still play important role in the toxicity caused by rhein in consideration of the
extensive metabolism and potential immuneꢀrelated toxicity.
UGT catalyzed the conjugation of lipophilic compounds in all living organisms.
There are 4 families of UGTs expressed in humans and other rodents: UGT1, UGT2,
UGT3 and UGT8, among which UGT2B7 is the most abundant UGT in human liver
(47). Liver appears to be the major site of UGT distribution, but some UGT those
isoforms found only in extrahepatic tissues, such as UGT1A7, 1A8 and 1A10 (48).
Our study revealed that the activity and positional preference of glucuronidation of
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rhein varied among different UGT isoforms. RG3 formation was mediated
predominantly by UGT 1A1, 1A9 and 2B7 and UGT1A1 showed the highest activity,
followed by 1A9 and 2B7, towards RG3 formation. Thus, these UGTs may play an
important role in both hepatic firstꢀpass elimination of rhein in human body. UGT
1A8 is the most potent isform mediating both RG1 and RG2 formation, which
specifically expressed in the gastrointestinal tract, indicating that the phenolic
hydroxyl groups of rhein predominantly underwent glucuronidation in the
gastrointestinal tract. It is worthy to note that UGT 2B7 showed the ability to solely
catalyse the glucuronidation of the carboxyl group in rhein, advising the advantage of
using UGT 2B7 expressed cell to further investigate the metabolic activation of rhein.
In conclusion, the present study confirmed that rhein is metabolized to acyl
glucuronide in human beings and the rhein acyl glucuronide is chemically reactive
and cytotoxic. There are significant species differences in the activation of rhein
between laboratory animals and human beings. The metabolic activation of rhein to its
acyl glucuronide might be the underlying mechanism of toxicity caused by
rheinꢀcontaining products.
ABBREVIATIONS
HepG2, hepatocarcinoma cell line; UGT, UDP glucuronosyltransferase; UDPGA,
uridine 5’ꢀdiphosphoglucuronic acid; HPLC, high performance liquid
chromatography; HLM, human liver microsomes; TMS, tetramethylsilane; GSH,
glutathione; MTT, 3ꢀ （ 4,5ꢀdimethylꢀ2ꢀthiazolyl)ꢀ2,5ꢀdiphenylꢀ2ꢀHꢀtetrazolium
390
391
392
393
394
bromide; IS, internal standard; PBS, phosphate buffer; RG1 and RG2, rhein hydroxyl
glucuronide; RG3, rhein acyl glucuronide
AUTHOR INFORMATION
Corresponding Author
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Dr. Jianqing Ruan, Soochow University, College of Pharmaceutical Sciences, Suzhou,
215123, People’s Republic of China. (Tel: +86 512 65882089; Fax: +86 512 65882089;
Eꢀmail: ruanjianqing@suda.edu.cn)
Dr. Hongjian Zhang, Soochow University, College of Pharmaceutical Sciences, Suzhou,
215123, People’s Republic of China. (Tel: +86 512 65882659; Fax: +86 512 65882089;
Eꢀmail: zhanghongjian@suda.edu.cn)
Notes
The authors declare no competing financial interest.
Funding
The present studies were supported by Jiangsu Province Science Foundation for
Youths (BK20150349).
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FIGURE CAPTIONS
Figure 1. Chemical structures of rhein, RG3 and hyperoside (A) and synthetic method
of RG3 (B).
Figure 2. MRM chromatograms of incubated samples of rhein with human liver
microsomes for 40 min (A), formation rates of glucuronides of rhein by human, rat,
mouse, monkey or dog liver microsomes (B) and typical MS1 and MS2 spectra of
rhein glucuronides (C). GlcA: glucuronic acid.
Figure 3. Stability of RG1, RG2 and RG3 in incubation solution (A), MRM
chromatograms incubation of RG3in PBS for 5 h (B) and stability of RG3 in PBS (C).
Figure 4. MRM chromatograms incubation of RG3 with GSH for 5 h (A), time course
of RG3 in the incubation with GSH (B) and MS spectra of RGSH (C). pGlu:
pyroglutamic acid.
Figure 5. Formation rates of RG1 and RG2 (A) and RG3 (B) by incubation of rhein
with recombinant UGTs for 40 min.
Figure 6. Effects of rhein concentration on formation of RG1, RG2 and RG3 by
different recombinant human UGTs.
Figure 7. Effects of different concentration of rhein and RG3 on the viability of
HepG2 cells. *** p<0.05 compared with vehicle control.
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Table 1 Kinetics Parameters of Rhein Glucuronidation in HLMs and Recombinant
Human UGTs.
RG3
RG2
RG1
Vmax
Clint
Km
(ꢁM)
Km
Km
(pmol/min/mg
protein)
(ꢁl/min/mg
(ꢁM)
(ꢁM)
protein)
HLMs
352.00 ± 13.12
82.04 ± 4.25
—
49.68 ± 3.85 7.10 ± 0.27 52.20 ± 5.96
14.81 ± 2.29 5.61 ± 1.00 13.79 ± 2.55
77.37 ± 9.98
26.10 ± 4.35
29.78 ± 2.35
80.00 ± 17.46
24.37 ± 3.01
—
UGT 1A1
UGT 1A7
UGT 1A8
UGT 1A9
UGT 2B7
—
—
—
—
25.12 ± 2.88
—
69.20 ± 14.95
70.96 ± 2.63
48.54 ± 3.37
20.86 ± 2.10 3.40 ± 0.55 15.36 ± 1.43
66.77 ± 8.73 0.73 ± 0.10
—
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Figure 1
A
B
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Figure 2
B
A
RG1
RG2
RG3
70
I.S.
2.2e4
2.0e4
1.8e4
1.6e4
1.4e4
1.2e4
1.0e4
8000.0
6000.0
4000.0
2000.0
0.0
60
50
40
30
20
RG3
10
RG2
rhein
RG1
5.70
0
1.0
2.0
3.0
4.0
5.0
6.0
Human liver microsomes
C
-Q1
[M-H-GlcA]-
282.9
-MS2 (459.00)
3.0e4
MS2
409.0
3.5e5
MS1
2.6e4
2.2e4
1.8e4
1.4e4
1.0e4
6000.0
2000.0
175.1
2.5e5
1.5e5
5.0e4
[M-H]-
459.1
513.3
459.1
489.5
400 420 440 460 480 500 520 540 560 580 600
m/z, Da
100
200
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m/z, Da
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Journal of Agricultural and Food Chemistry
Figure 3
RG1
RG2
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150
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60
30
0
A
0
5
10
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Time(hr)
I.S.
16
0.6
0.4
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0.0
RG3
2.8e4
2.4e4
B
C
Rhein
RG4
12
8
2.0e4
1.6e4
1.2e4
RG4
RG3
8000.0
4000.0
4
rhein
0
0.0
1.0
2.0
3.0
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0
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Time, min
Time(hr)
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