Comparative Study of Hepatotoxicity of Pyrrolizidine Alkaloids Retrorsine and Monocrotaline

Article abstract of DOI:10.1021/acs.chemrestox.6b00260

Many pyrrolizidine alkaloids (PAs) can cause liver injury in animals and humans. Different hepatotoxic PAs can produce similar hepatotoxic effects, but the degree of their toxicities may vary widely. Retrorsine (RTS) and monocrotaline (MCT) share the same core structure (retronecine) and similar metabolic activation pathway. RTS and MCT both produced liver injury, but the former was more hepatotoxic than the latter. Enzyme kinetic study demonstrated that the value of V_max/K_m for RTS was 5.5-fold larger than that of MCT. Additionally, RTS produced higher levels of pyrrole-glutathione (GSH) conjugates and protein covalent binding than MCT at the same dose. Furthermore, RTS induced significant hepatic GSH depletion but MCT did little. This comparative study provides clear evidence that the generation of the reactive pyrrolic intermediates plays a critical role in PA-induced hepatotoxicity.

Full text of DOI:10.1021/acs.chemrestox.6b00260

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Article
A Comparative Study of Hepatotoxicity of
Pyrrolizidine Alkaloids Retrorsine and Monocrotaline
Xiaojing Yang, Weiwei Li, Ying Sun, Xiucai Guo, Wenlin Huang, Ying Peng, and Jiang Zheng
Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00260 • Publication Date (Web): 17 Jan 2017
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A Comparative Study of Hepatotoxicity of Pyrrolizidine Alkaloids Retrorsine
and Monocrotaline
Xiaojing Yang†, Weiwei Li†, Ying Sun†, Xiucai Guo†, Wenlin Huangθ, Ying Peng†*, and Jiang
§
*
Zheng†¶
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†Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang, Liaoning, 110016, P. R.
China
θDepartment of Biochemistry, University of Washington, Seattle, WA 98195， USA
¶Key Laboratory of Pharmaceutics of Guizhou Province, §State Key Laboratory of Functions and
Applications of Medicinal Plants, Guizhou Medical University, Guiyang, Guizhou, 550004, P. R.
China
Running title:
Hepatotoxicity of retrorsine and monocrotaline
*Correspondence authors
Jiang Zheng
Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang,
Liaoning, 110016, P. R. China; Key Laboratory of Pharmaceutics of Guizhou
Province, State Key Laboratory of Functions and Applications of Medicinal Plants,
Guizhou Medical University, No.9, Beijing Road, Yunyan District, Guiyang 550004,
China
Eꢀmail: zhengneu@yahoo.com
Tel: +86ꢀ24ꢀ23986361
Fax: +86ꢀ24ꢀ23986510
Ying Peng
Wuya College of Innovation, Shenyang Pharmaceutical University, 103 Wenhua Rd,
Shenhe Qu, Shenyang Shi, Liaoning Sheng, China, 110016 Eꢀmail:
yingpeng1999@163.com
Tel: +86ꢀ24ꢀ23986361
Fax: +86ꢀ24ꢀ23986510
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Abbreviations: ALT, alanine transaminase; AST, aspartate transaminase; AUC, area
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under curve; CE, collision energy; CXP, cell exit potential; dehydroꢀMCT,
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dehydromonocrotaline;
DP,
declustering
potential;
DABA:
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4ꢀdimethylaminobenzaldehyde; dehydroꢀRTS, dehydroretrorsine; ESI, electrospray
ionization; EP, entrance potential; GSH, glutathione; IS, internal standard; i.p.,
intraperitoneally; LCꢀMS/MS, liquid chromatography coupled to tandem mass
spectrometry; MCT, monocrotaline; mBrB, monobromobimane; MRM,
multipleꢀreaction monitoring；MRT, mean residence time; NADPH, βꢀnicotinamide
adenine dinucleotide 2’ꢀphosphate reduced tetrasodium salt; PAs, pyrrolizidine
alkaloids; RTS, retrorsine.
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Table of Contents (TOC) graphic
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800
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**
MCT
RTS
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*
Monocrotaline
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（MCT）
P450
Hepatotoxicity
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MCT
RTS
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Retrorsine
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（RTS）
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ABSTRACT
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Many pyrrolizidine alkaloids (PAs) can cause liver injury in animals and
humans. Different hepatotoxic PAs can produce similar hepatotoxic effects, but the
degree of their toxicities may vary widely. Retrorsine (RTS) and monocrotaline
(MCT) share the same core structure (retronecine) and similar metabolic activation
pathway. RTS and MCT both produced liver injury but the former was more
hepatotoxic than the latter. Enzyme kinetic study demonstrated that the value of
Vmax/Km for RTS was 5.5ꢀfold larger than that of MCT. Additionally, RTS
produced higher levels of pyrroleꢀGSH conjugates and protein covalent binding than
MCT at the same dose. Furthermore, RTS induced significant hepatic GSH
depletion but MCT did little. This comparative study provided clear evidence that
the generation of the reactive pyrrolic intermediates plays a critical role in
PAꢀinduced hepatotoxicity.
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INTRODUCTION
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Pyrrolizidine alkaloids (PAs) are common constituents of hundreds of plant
species of various unrelated botanical families distributed in many geographical
regions of the world.1ꢀ3 It was reported that toxic PAs (mainly referring those with
1,2ꢀunsatuation) were found in about 3% of the world’s flowering plants and over
6,000 plants, and about half of them exhibit toxic activities.4,5 PAꢀcontaining plants
are probably the most common poisonous plants affecting livestock, wildlife, and
humans. Humans could be exposed to these hepatotoxic PAs through consumption
of contaminated grains,6,7 herbal preparations,8 herbal tea9 or dietary components,
such as milk and honey.10 PAs were found to exhibit acute toxicity and chronic
toxicity. Acute liver injury includes hemorrhagic necrosis, hepatic megalocytosis,
venoꢀocclusion, liver cirrhosis, nodular hyperplasia and hepatic carcinomas.11,12
Toxicities resulting from chronic exposure to PAs occur mainly in liver, lungs, blood
vessels, and in some instances kidneys, pancreas, gastrointestinal tract, bone marrow,
and brain.13
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According to the necine bases in their chemical structures, PAs are divided into
four main types: otonecine, retronecine, heliotridine (the enantiomer of retronecine
the Cꢀ7 position), and platyphylline. The most of toxic PAs are those belonging to
14ꢀ16
the first three types that contain a double bond in the base
.
Both retrorsine
and monocrotaline (MCT) belong to the category of retronecineꢀtype of PAs. RTS
a 12ꢀmembered macrocyclic diꢀester pyrrolizidine alkaloid with an ꢀunsaturated
α, β
double bond linked to the ester group at the C7 position of the necine base and exists
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Senecio spp. at different regions of the world.2 MCT, an 11ꢀmembered macrocyclic
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diꢀester without
α, βꢀunsaturated double bond, occurs in plants of the genus
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Crotalaria17 and has been used as a model compound to induce pulmonary
hypertension. In general, there are three principal pathways for PA metabolism,
including hydrolysis of PAs to release necines and necic acids, Nꢀoxidation to form
Nꢀoxides, and oxidation of PAs to produce diꢀdehydroꢀpyrrolizidine (pyrrolic esters)
intermediates. The first two are regarded as detoxication pathways. Metabolic
activation of hepatotoxic PAs, involving the formation of reactive pyrrolic
intermediates (pyrrolic esters), has been considered as the key step for PAꢀinduced
toxicities.18ꢀ21 These pyrrolic intermediates are electrophilic species and chemically
reactive. Once formed, they can bind to nucleophilic biomolecules, such as one or
two molecules of glutathione to form glutathione conjugates17,22,23 or react with
and proteins to generate DNA and protein adducts.23,24 RTS and MCT are
bioactivated through the similar metabolic pathway as above, and the resulting
adducts derived from their pyrrole metabolites share the same pyrrole core (Scheme
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Compared to MCT, the
α, βꢀunsaturation of RTS in the acid moiety may enhance
steric hindrance around the Cꢀ7 ester group to limit its hydrolysis.13 And this group
also responsible for higher lipophilicity that facilitates metabolism. Since the levels
of PAs and their metabolites in the body would change over time, and the patterns of
their toxicodynamics and toxicokinetics need to be addressed to better understand
mechanisms of PA toxic actions. In addition, hepatic toxicity is a complex
of several pathophysiological mechanisms. The effects of hepatotoxicity induced
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different PAs are most likely similar, but the degree of their hepatotoxicity may vary.
The LD50 values for RTS, MCT, senecionine, and seneciphylline in male rats were
reportedly 34, 109, 50, and 77 mg/kg respectively, after a single intraperitoneal
injection.13 Toxicokinetic behavior, potential to deplete hepatic GSH, protein
modifications, GSH conjugation, and metabolic activation efficiency are the
possibly involved in the determination of the toxicity potentials of PAs. The major
objective of the present study was to define the roles of such factors in metabolic
activation and hepatotoxicities of MCT and RTS. The comparative study may
us to better understand the mechanisms of PA toxic action.
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EXPERIMENTAL PROCEDURE
Chemicals
Monocrotaline (MCT, ≥98%), retrorsine (RTS,
glutathione (GSH, 98%), Sꢀhexylglutathione (>97%), silver nitrate,
≥
98%), diazepam (≥98%),
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≥
monobromobimane (mBrB, >97%), 4ꢀdimethylaminobenzaldehyde (DABA),
5ꢀsulfosalicylic acid, and NADPH were purchased from Sigma Chemical Co. (St.
Louis, MO). Formic acid, trifluoroacetic acid, and perchloric acid were purchased
from Fisher Scientific (Springfield, NJ). All organic solvents were got from Fisher
Scientific (Springfield, NJ). All reagents and solvents were either analytical or
HPLC grade.
Animals and treatment
Male Kunming mice (18ꢀ20 g) were purchased from the Animal Center of
Shenyang Pharmaceutical University (Shenyang, China). Mice were allowed free
access to food and water and were housed in a temperatureꢀcontrolled (22±4 °C)
facility with a 12 h dark/light cycle for at least 5 days after receipt and before
treatment. All animal studies were performed according to procedures approved by
the Ethics Review Committee for Animal Experimentation of Shenyang
Pharmaceutical University. Mice were randomly divided into three groups. One
group containing fifteen mice was treated intraperitoneally (i.p.) with corn oil as the
vehicle group. The blood and liver were harvested at 4, 8, 12, 24 and 36 h after the
administration (n=3 each time point). The other groups containing twentyꢀseven
mice were treated intraperitoneally (i.p.) with MCT or RTS suspended in corn oil at a
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dosage of 0.2 mmol/kg (MCT: 65 mg/kg, RTS: 70 mg/kg), respectively. The dose
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applied, lower than LC50 and higher than that (20 µmol/kg) reported in a study,25
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based on our preliminary study that showed appropriate degrees of hepatotoxicity
GSH depletion induced by RTS and MCT.
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The mice were weighted and then euthanized (one mouse each time). Blood
and liver were harvested at 10 min, 30 min, 1, 2, 4, 8, 12, 24 and 36 h (n=3 each time
point) after the administration. The liver tissues (0.2 g) were homogenized in 2 mL
phosphate buffer (pH 7.4, 0.1 M), followed by centrifuging at 9,000 g for 10 min.
The supernatants were stored at ꢀ80 °C until analysis. Plasma samples were
harvested for determination of GSH conjugates and GSH contents, and serum
samples were collected for aspartate transaminase (AST) and alanine transaminase
(ALT) assays on VITROS® 5600 Integrated System (OrthoꢀClinical Diagnostics,
Rochester, NY). They were freshly prepared from each blood sample collected,
and all specimens were stored at ꢀ80 °C until analysis.
Assessment of GSH
Quantitative analyses of hepatic GSH levels were performed by following the
procedure developed by our laboratory.26 Briefly, the liver supernatants (60
GSHꢀcontaining solution were mixed with a monobromobimane (mBrB) solution
mg/mL in acetonitrile, 20 L). The resulting mixtures were allowed to stand at
ꢀL) or
ꢀ
temperature in the dark for 20 min to produce mBrBꢀderived GSH conjugate
2), followed by addition of 10 µL of Sꢀhexylglutathione (58.8 ng/mL) as the internal
standard (IS) and of 100 ꢀL 10% 5ꢀsulfosalicylic acid to precipitate protein. The
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mixtures were centrifuged at 13,000 g for 10 min, and the supernatants were injected
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into a LCꢀMS/MS system for analysis.
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Toxicokinetic studies of RTS and MCT
The plasma levels of RTS and MCT were estimated by modification of a
reported method.27 To 20 µL of plasma sample was a 10 µL aliquot of IS added,
followed by vortexing for 10 s. The resulting sample was mixed with 2 volumes of
acetonitrile and centrifuged at 19,000 g for 10 min to remove protein. The
supernatants were dissolved with 3 volumes of water and then subjected to the
LCꢀMS/MS system for analysis.
Determination of pyrrole-GSH conjugates
The determination of pyrroleꢀGSH conjugates was conducted, according to a
published procedure.24 An aliquot of 80 µL acetonitrile containing 12.5 ng
Sꢀhexylglutathione used as IS was added to 40 µL of the plasma or liver samples.
After centrifugation at 14,000 g for 5 min, the resulting supernatants were diluted
with 3 volumes of water and then injected (5.0 ꢀL) into the LCꢀMS/MS for analysis.
Determination of pyrrole-protein adduction
Liver pyrroleꢀprotein adduction levels were determined using the method
established in our laboratory.12 Briefly, liver homogenates were mixed with 5
volumes of acetone, vortexed, and centrifuged at 900 g for 5 min. After washing
absolute ethanol, the resulted pellets were reconstituted in 2% silver nitrate ethanol
solution containing 5% trifluoroacetic acid and were shaken for 30 min at room
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temperature. After centrifugation at 19,000 g for 10 min, the resulting supernatants
were reacted with 4ꢀdimethylaminobenzaldehyde (v/v 4:1) in ethanol containing 1%
perchloric acid at 55 °C for 10 min (Scheme 3). The resultant mixtures were mixed
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with 10 µL IS solution (diazepam, 1.25 ꢀg/mL) and injected into the LCꢀMS/MS
system for analysis.
In vitro microsomal metabolism of MCT and RTS
Mouse (male) liver microsomes were prepared as described by our laboratory.28
The incubation mixtures contained 0.25 mg/mL mouse liver microsomal protein, 3.2
mM MgCl2, 10 mM GSH, and MCT or RTS (5ꢀ2,000 µM) in a final volume of 500
µL. The reaction was initiated by addition of NADPH (final concentration: 1.0 mM)
and stopped by addition of 500 µL iceꢀcold acetonitrile after 20 min incubation at
37 °C. The resulting mixtures were vortexꢀmixed and centrifuged at 19,000 g for
10 min. The supernatants were dissolved with 3 volumes of water and then
analyzed by the LCꢀMS/MS system.
LC-MS/MS method
The quantification of MCT, RTS and their metabolites were performed on an
SCIEX Instruments 5500 triple quadrupole mass spectrometry (Applied Biosystems,
Foster City, CA) equipped with an Agilent 1260 Series Rapid Resolution LC system.
Data were analyzed using Applied Biosystems/SCIEX AnalystTM software (version
1.6.2). Samples were analyzed by multipleꢀreaction monitoring (MRM) scanning
positive ion mode. The optimized MS instrument parameters obtained after tuning
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were as follows: ion spray voltage was set at 5,500 V, and ion source temperature
set at 650 °C. Curtain gas, gas 1, and gas 2 were 20, 50, and 50 psi, respectively.
The characteristics of ion pairs (declustering potential, DP; collision energy, CE; and
collision cell exit potential, CXP) and respective LC conditions were listed as
For analyses of MCT and RTS, chromatographic separations were achieved on
a CAPCELL PAK UGꢀC18 column (250 × 4.6 mm, 5.0 µm) (Shiseido, Tokyo, Japan)
protected by a SecurityꢀGuard (4.0 × 3.0 mm, 5.0 µm) C18 column (Phenomenex,
Torrance, CA) which were also used for the analyses of pyrroleꢀGSH conjugates and
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pyrroleꢀprotein
adduction.
Mobile
phase
was
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acetonitrile:water:formic acid (v/v/v=15:85:0.1). Flow rate was set at 0.8 mL/min.
The characteristic ion pairs (DP, CE, and CXP) for MCT and RTS were m/z
326.1→120.2 (270, 34, 13) and m/z 352.1→119.9 (100, 48, 10), respectively. MCT
and RTS acted as IS for each other.
For detection of GSH,26 chromatographic separation was performed on a BDS
HYPERSIL C18 ODS column (5.0 ꢀm, 150 mm × 4.6 mm; Thermo Fisher, San Jose,
CA) at 25 °C, using 0.1% formic acid in acetonitrile and in water as mobile phases A
and B, respectively. The employed gradient eluting program was performed as
follows: 0ꢀ1 min, 90ꢀ90% B; 1ꢀ6 min, 90ꢀ10% B; 6ꢀ9 min, 10ꢀ90% B; 9ꢀ10 min,
90ꢀ90% B. Flow rate was 1.0 mL/min. The characteristic ion pairs (DP, CE, and
CXP) were m/z 498.2→192.2 (115, 50, 3) for GSHꢀmBrB and m/z 392.2→246.3 (86,
24, 5) for Sꢀhexylglutathione (IS), respectively.
For determination of pyrroleꢀGSH conjugates, mobile phase system containing
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acetonitrile with 0.1 % formic acid (A) and water with 0.1 % formic acid (B) was
with a gradient elution as follows: 0ꢀ2 min, 90ꢀ90 % B; 2ꢀ10 min, 90ꢀ10 % B; 10ꢀ12
min, 10ꢀ10 % B; 12ꢀ14 min, 10ꢀ90 % B; and 14ꢀ17 min, 90ꢀ90 % B. Flow rate was
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0.8 mL/min.
The characteristic ion pairs (DP, CE, and CXP) were m/z
(100, 30, 13) for GSH conjugate and m/z 392.2→246.3 (86, 24, 5) for
Sꢀhexylglutathione (IS), respectively.
For analysis of pyrroleꢀprotein adduction, mobile phase systems containing
acetonitrile with 0.1 % formic acid (A) and water with 0.1 % formic acid (B) were
used with a gradient elution as follows: 0ꢀ2 min, 70ꢀ70 % B; 2ꢀ10 min, 70ꢀ10 % B;
10ꢀ12 min, 10ꢀ10 % B; 12ꢀ14 min, 10ꢀ70 % B; and 14ꢀ17 min, 70ꢀ70 % B. Flow
rate was set at 0.8 mL/min. The characteristic ion pairs (DP, CE, and CXP) were
m/z 341.0→252.1 (100, 40, 10) for pyrroleꢀprotein adduction and m/z 285.1→193.5
(101, 46, 10) for diazepam (IS), respectively.
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RESULTS
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Hepatotoxicity of RTS and MCT
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Both RTS and MCT caused elevations of serum AST and ALT activities in a
timeꢀdependent manner in mice after intraperitoneal administration. At the dose of
0.2 mmol/kg, the increases in serum AST and ALT activities continued until 36 h
post treatment (Fig. 2). At 36 h, serum ALT and AST activities of RTSꢀtreated
mice were 568.0±105.1 (U/L) and 310.3±44.7 (U/L) respectively, over 3ꢀfold higher
than that of MCTꢀtreated animals (p<0.05). In addition, a significant increase of the
relative weights of liver (the ratio of organ weight vs. body weight) was observed in
RTSꢀtreated mice at 36 h (p<0.05), compared with that of vehicleꢀtreated animals
(Fig. 1). Apparently, there was negligible difference in the values of ALT, AST, and
the relative weights of liver between MCTꢀ and vehicleꢀtreated mice.
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Hepatic GSH depletion induced by RTS and MCT in mice
Time course hepatic GSH contents were monitored to assess the efficiency of
metabolic activation of RTS and MCT as well as the electrophilicity of their reactive
metabolites. The typical chromatogram of GSHꢀmBrB is shown in Fig. 3C.
Hepatic GSH levels decreased and reached the lowest level (14 % of the levels at
time 0 h) from 10 min to 30 min after a hepatotoxic dose (0.2 mmol/kg) of RTS (Fig.
5). In contrast, no such GSH depletion was observed in mice given equiꢀmolar
dose of MCT.
Toxicokinetic behaviors of RTS and MCT
The levels of the parent PAs in plasma were monitored at various time points
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after treatment (i.p.) with 0.2 mmol/kg MCT or RTS. The representative
chromatograms of MCT and RTS are shown in Fig. 3A and 3B, the mean plasma
concentrationꢀtime curves of MCT and RTS are presented in Fig. 4, and the main
toxicokinetic parameters are listed in Table 1. Apparently, little difference in
MRT and CLz/F was observed in mice treated with RTS or MCT. However,
increased Cmax (1.7ꢀfold), Vz/F (2.2ꢀfold), and t1/2 (2ꢀfold) were observed in mice
treated with RTS, compared with those in mice treated with MCT.
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Pyrrole-GSH conjugates in plasma and liver of mice given RTS or MCT
Both MCT and RTS were metabolized to the corresponding pyrrolic esters, i.e.
dehydroꢀmonocrotaline (dehydroꢀMCT) (3) and dehydroꢀretrorsine (dehydroꢀRTS)
(4), respectively (Scheme 1). The reactive metabolites sequentially reacted with
GSH to form GSH conjugates. As expected, three peaks responsible for
monoꢀGSH conjugates (6 and 7, Scheme 1) were detected by LCꢀMS/MS, and Fig.
3D shows the representative chromatograms of the GSH conjugates. Timeꢀcourse
of hepatic and plasma GSH conjugates 6 and 7 were monitored in mice treated with
RTS or MCT. A 2ꢀfold increase in the level of hepatic GSH conjugates 6 and 7
(integrated) was observed in mice given RTS relative to that in animals treated with
MCT. Furthermore, the Cmax and AUC(0ꢀt) of plasma GSH conjugates 6 and 7 in
mice treated with RTS were 6ꢀfold and 2.9ꢀfold higher than those in animals given
MCT, respectively (Fig. 6).
Pyrrole-derived protein modification in liver of mice treated with RTS or MCT
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The levels of hepatic pyrroleꢀprotein adduction in mice given RTS or MCT
were assessed by chemical derivatization of the protein adduct to compound 12
(Scheme 3). The representative chromatogram of compound 12 is shown in Fig.
3E. RTSꢀ and MCTꢀinduced hepatic protein adduction reached the peak at 2 and 1
h respectively after a single dose (i.p.) of the individual alkaloids (Fig. 7).
Interestingly, 5.3ꢀ (Cmax) and 3.8ꢀfold (AUC0ꢀt) elevations of the protein adduction
were observed in the liver of the mice given RTS, compared with that of the animals
treated with the same molar dose of MCT.
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Metabolic activation efficiency of RTS and MCT in mouse liver microsomes
Enzyme kinetic parameters of metabolic activation of RTS and MCT were
determined by microsomal incubation with RTS or MCT at a serial of concentrations
(5ꢀ2,000 µM). Km and Vmax values were calculated based on the production of
GSH conjugates 6 and 7 (integrated). The MichalisꢀMenten plots were constructed
and are shown in Fig. 8, and the calculated parameters are summarized in Table 2.
Apparently, Km value for MCT was higher than that for RTS (642.0±44.6 vs.
278.5±20.8), and Vmax value of MCT was less than that of RTS (24.45±0.68 vs.
58.06±2.82). Vmax/Km known as intrinsic clearance (CLint), an indicator of the
efficiency of enzyme catalysis, was calculated, and the value of CLint for RTS was
about 5.5ꢀfold higher than that of MCT.
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DISCUSSION
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Earlier studies have revealed that PAs with a broad range of structure show
similar hepatotoxic effects, but the intensity of their toxicities differed widely.17
This would be a good probe to gain the insight into the mechanisms of PAꢀinduced
toxicities. We started with comparing the hepatotoxicity of RTS and MCT which
both belong to the category of retronecineꢀtype PAs and found that RTS induced
intensified hepatotoxicity than MCT in mice. RTS exhibited 10.3ꢀ and 3.6ꢀfold
higher serum ALT and AST activities than MCT in animals given the same molar
dose (0.2 mmol/kg, i.p.) 36 h after administration. The treatment of RTS or MCT
caused elevated relative weight of livers in mice, compared with that of animals
treated with vehicle. The observed increase in relative weight of liver may result
from expanded sinusoids due to congestion and edema arising from accumulation of
fluids and red blood cells within hepatic cords.29
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Importantly, mice treated with RTS received significant gain (p<0.05) in
relative weight of liver 36 h post dosing, compared with that of animals given the
same dose of MCT. The observation provided further evidence that RTS was more
hepatotoxic than MCT.
The observed difference in intensity of liver toxicity induced by RTS and MCT
offers a unique starting point for the mechanistic studies of PA toxicity. As the
step, we evaluated the toxicokinetic behaviors of the two alkaloids. Apparently,
and MCT revealed similar AUC (Table 1), indicating the two alkaloids shared
toxicokinetic behavior. Although Cmax of RTS was found to be 1.67ꢀfold higher
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that of MCT, it is unlikely that such increase in Cmax would cause the dramatic
elevations in serum ALT and AST activities observed.
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The working hypothesis of the present study was that pyrrolic esters play a
critical role in PAꢀinduced toxicities. We reasoned that the amounts of pyrrolic
generated and their chemical reactivity (electrophilicity) towards protein are both
critical for toxicities of PAs and that the PAs metabolized to reactive metabolites
higher efficiency are more toxic than those with poor effectiveness of metabolic
activation. Enzyme kinetic parameters Vmax and Km of RTS and MCT were
determined by monitoring the formation of GSH conjugates 6 and 7 in mouse liver
microsomes fortified with GSH. The value of CLint (Vmax/Km) of RTS was 5.5ꢀfold
greater than that of MCT, indicating the metabolic activation of RTS in mice was
effective than that of MCT. This can explain the observation that RTS was more
hepatotoxic than MCT in mice. It should be mentioned that the obtained parameter
CLint reflected not only the efficiency of microsomal bioactivation of the two
but also the reactivity (electrophilicity) of their reactive metabolites. The
electrophilicity intensity of the reactive metabolites can be another factor possibly
playing a critical role in PA toxicities. Unfortunately, we are unable to determine
electrophilicities of dehydroꢀRTS (4) and dehydroꢀMCT (3) due to their instability,
although we successfully synthesized the two reactive metabolites. Otherwise, we
would have identified the contribution of the reactivity of the two reactive
to the values of CLint obtained. MCT has been known to be more pneumotoxic but
less hepatotoxic than RTS and many other PAs. An explanation is that
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is relatively more stable than dehydroꢀRTS and escapes from the liver and migrates
the lung. However, other studies showed that the halfꢀlife of dehydroꢀMCT ranged
from 3 to 5 sec in aqueous solution,30,31 suggesting that the reactive intermediate is
that stable. Thus, a comparison study on reactivities of dehydroꢀMCT and
dehydroꢀRTS is needed to better understand the mechanisms of MCTꢀinduced
pneumotoxicity, although it is a challenging study. GSH conjugation with the
reactive metabolites of RTS and MCT was evident in vitro. GSH conjugates 6 and
were also detected in plasma and liver tissues of animals given RTS or MCT. The
concentrations of plasma and hepatic GSH conjugates 6 and 7 in mice treated with
RTS were 2.9ꢀfold and 2.7ꢀfold higher than those in animals given MCT (Fig. 6).
This was consistent with the observation of the greater value of CLint for RTS than
for MCT obtained in the microsomal incubation study. As a result, the GSH
conjugation led to consumption of hepatic GSH and could result in GSH depletion.
Depleted cellular GSH content has been generally accepted as a biomarker of early
stage of cell injury. A study showed that infusion of glutathione into the portal
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circulation prevented sinusoidal obstruction syndrome caused by MCT in vivo.29
A
single dose of RTS induced significant depletion of hepatic GSH in mice, while the
same molar dose of MCT elicited little effect on hepatic GSH levels (Fig. 5). The
findings, along with the abundance of GSH conjugates 6 and 7 found in plasma and
liver of RTSꢀtreated mice, provide additional evidence in agreement with the
observation of intensified hepatotoxicity induced by RTS.
Protein covalent binding by reactive metabolites is an important mechanism of
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toxic action.12 The present study showed that RTS produced 3.8ꢀfold higher
pyrroleꢀprotein adduction than MCT did at the same dose (Fig. 7). The observation
of high volume protein adduction induced by RTS provided us with better
for the observed difference in the intensity of hepatotoxicity induced by RTS and
The combined findings led us to propose that the effective metabolic activation of
may be an important factor which determines the intensity of intrinsic toxicity of
Specifically, the observed larger quantity of dehydroꢀRTS (4) generated from
P450ꢀmediated metabolic activation of RTS reacted with GSH resulted in more
production of GSH conjugates 6ꢀ7 and intensified GSH depletion. Additionally, the
reactive species modified hepatic protein to form protein covalent binding, and
modification possibly initiates cytotoxicity.
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This study facilitated the understanding of the mechanisms of PA toxic action.
Although the data on hepatotoxicity,13 metabolic activation efficiency,32 production
of GSH conjugate,33 and protein adduction25 induced by RTS and MCT are available,
these studies were carried out in either mice, rats, or humans by different
independent laboratories, which makes comparison in parallel difficult. The
present study systematically compared body weight gain, GSH depletion,
toxicodynamics, and toxicokinetics, along with the above parameters. Additionally,
the work was performed in the same type of animals in the same laboratory. The
results provided a comprehensive and more convincing picture of toxic actions of
RTSIanncdoMncCluTs.ion, RTS was more hepatotoxic than MCT, along with indications of
more effectiveness of metabolic activation, more production of the corresponding
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GSH conjugates, intensified hepatic GSH depletion, and more protein covalent
binding. The high volume production of dehydroꢀRTS is likely one of important
factors that made RTS more hepatotoxic than MCT. This comparative study
us to define the critical steps responsible for the potency of hepatotoxicity induced
PAs.
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Funding sources
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This work was supported by the National Natural Science Foundation of China
Grant 81430086 and 81373471 (in part).
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formed from pyrrolizidine alkaloids by hepatic microsomes in vitro: Relevance to in
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vivo hepatotoxicity. Chem. Biol. Interact. 43, 209ꢀ222.
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of a mixed function oxidase inducer and inhibitor on monocrotaline pyrrole
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the hepatic toxicity and metabolism of Crotalaria assamica and Eupatorium species.
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Rader, J. I. (2012) Comparison of metabolismꢀmediated effects of pyrrolizidine
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glutathione conjugates. Xenobiotica 42, 1038–1048.
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Table 1. Summary of RTS and MCT toxicokinetic parametersa obtained in mice
after a single intraperitoneal injection of RTS or MCT (0.2 mmol/kg, n=3)
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Parameters
MCT
RTS
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Cmax (mg/L)
AUC(0ꢀt) (mg/L×h)
AUC(0ꢀ∞) (mg/L×h)
MRT(0ꢀt) (h)
t1/2 (h)
13.40±2.76
12.73±1.00
12.76±1.00
0.78±0.07
0.34±0.08
0.50
22.67±3.06
12.43±0.67
12.43±0.66
0.61±0.07
0.67±0.23
0.17
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44
45
46
47
48
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Tmax (h)
CLz/F (L/h/kg)
Vz/F (L/kg)
5.13±0.41
2.52±0.43
5.64±0.31
5.54±2.10
a. The toxicokinetic parameters were calculated, using Drug and Statistic (DAS) 2.1.1 software
(Mathematical Pharmacology Professional Committee of China, Shanghai, China). The time
points to collect blood samples were 10 min, 30 min, 1, 2, 4, 8, 12, 24 and 36 h (n=3 each time
point) after the administration.
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Table 2. Enzyme kinetic parametersa of RTS and MCT in mouse liver microsomes
after 20 min of incubation (final concentrations of RTS and MCT: 5ꢀ2,000 µM)
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Vmax
Km
R2
Compounds
Vmax/Km
( M)
ꢀ
(Area ratio/min/mg protein)
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MCT
RTS
24.45±0.68
58.06±2.82
642.0±44.6
278.5±20.8
0.038
0.208
0.99
0.99
a. The enzyme kinetic parameters were obtained using GraphPad Prism 5 (San Diego, CA, USA).
MCT or RTS (5ꢀ2,000 µM) were incubated with 0.25 mg/mL mouse liver microsomal protein in
the presence of NADPH and GSH. The catalytic capabilities of the enzymes were evaluated by
monitoring the formation of the pyrrolicꢀGSH conjugates. Each value represents the mean and
standard deviation of three experiments.
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