Comparative Study of Hepatotoxicity of Pyrrolizidine Alkaloids

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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 Downloaded from http://pubs.acs.org on January 23, 2017

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Chemical Research in Toxicology

A Comparative Study of Hepatotoxicity of Pyrrolizidine Alkaloids Retrorsine and Monocrotaline θ

Xiaojing Yang†, Weiwei Li†, Ying Sun†, Xiucai Guo†, Wenlin Huang , Ying Peng†*, and Jiang Zheng†¶§*

†

Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang, Liaoning, 110016, P. R. China θ

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

¶

Key Laboratory of Pharmaceutics of Guizhou Province, §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 E-mail: [email protected] Tel: +86-24-23986361 Fax: +86-24-23986510

Ying Peng Wuya College of Innovation, Shenyang Pharmaceutical University, 103 Wenhua Rd, Shenhe Qu, Shenyang Shi, Liaoning Sheng, China, 110016 E-mail: [email protected] Tel: +86-24-23986361 Fax: +86-24-23986510

ACS Paragon Plus Environment


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Abbreviations: ALT, alanine transaminase; AST, aspartate transaminase; AUC, area under curve; CE, collision energy; CXP, cell exit potential; dehydro-MCT, dehydromonocrotaline;

DP,

declustering

potential;

DABA:

4-dimethylaminobenzaldehyde; dehydro-RTS, dehydroretrorsine; ESI, electrospray ionization; EP, entrance potential; GSH, glutathione; IS, internal standard; i.p., intraperitoneally; LC-MS/MS, liquid chromatography coupled to tandem mass spectrometry;

MCT,

monocrotaline;

mBrB,

monobromobimane;

MRM,

multiple-reaction monitoring；MRT, mean residence time; NADPH, β-nicotinamide adenine dinucleotide 2’-phosphate reduced tetrasodium salt; PAs, pyrrolizidine alkaloids; RTS, retrorsine.


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Monocrotaline （ MCT））

Dehydromonocrotaline P450

OH O

O

O H

N

Retrorsine （ RTS））

** MCT

400

RTS

* 0 0

20 Time (h)

40

OH

OH

O

800

Hepatotoxicity

OH

O

AST activities (U/L)

Table of Contents (TOC) graphic

O O

O

400

ALT activities (U/L)

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**

200

N

Dehydroretrorsine

MCT

**

RTS 0 0


20 Time (h)

40


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ABSTRACT 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 5.5-fold larger than that of MCT.

Additionally, RTS

produced higher levels of pyrrole-GSH conjugates and protein covalent binding than MCT at the same dose. depletion but MCT did little.

Furthermore, RTS induced significant hepatic GSH This comparative study provided clear evidence that

the generation of the reactive pyrrolic intermediates plays a critical role in PA-induced hepatotoxicity.


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INTRODUCTION Pyrrolizidine alkaloids (PAs) are common constituents of hundreds of plant species of various unrelated botanical families distributed in many geographical regions of the world.1-3

It was reported that toxic PAs (mainly referring those with

1,2-unsatuation) 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

PA-containing 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 toxicity.

PAs were found to exhibit acute toxicity and chronic

Acute liver injury includes hemorrhagic necrosis, hepatic megalocytosis,

veno-occlusion, 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 According to the necine bases in their chemical structures, PAs are divided into four main types: otonecine, retronecine, heliotridine (the enantiomer of retronecine the C-7 position), and platyphylline.

The most of toxic PAs are those belonging to

the first three types that contain a double bond in the base.14-16 Both retrorsine and monocrotaline (MCT) belong to the category of retronecine-type of PAs.

RTS

a 12-membered macrocyclic di-ester pyrrolizidine alkaloid with an α, β-unsaturated 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 11-membered macrocyclic di-ester without α, β-unsaturated double bond, occurs in plants of the genus 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, N-oxidation to form N-oxides, and oxidation of PAs to produce di-dehydro-pyrrolizidine (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 PA-induced toxicities.18-21 reactive.

These pyrrolic intermediates are electrophilic species and chemically

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 Compared to MCT, the α, β-unsaturation of RTS in the acid moiety may enhance steric hindrance around the C-7 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, ≥98%), diazepam (≥98%), glutathione

(GSH,

monobromobimane

≥

98%),

(mBrB,

S-hexylglutathione

>97%),

(>97%),

silver

4-dimethylaminobenzaldehyde

nitrate, (DABA),

5-sulfosalicylic 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). Scientific (Springfield, NJ).

All organic solvents were got from Fisher

All reagents and solvents were either analytical or

HPLC grade.

Animals and treatment Male Kunming mice (18-20 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 temperature-controlled (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 twenty-seven

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

applied, lower than LC5013 and higher than that (20 µmol/kg) reported in a study,25 based on our preliminary study that showed appropriate degrees of hepatotoxicity GSH depletion induced by RTS and MCT. 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 (Ortho-Clinical 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 µL) or GSH-containing solution were mixed with a monobromobimane (mBrB) solution mg/mL in acetonitrile, 20 µL).

The resulting mixtures were allowed to stand at

temperature in the dark for 20 min to produce mBrB-derived GSH conjugate 2), followed by addition of 10 µL of S-hexylglutathione (58.8 ng/mL) as the internal standard (IS) and of 100 µL 10% 5-sulfosalicylic acid to precipitate protein.


The


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mixtures were centrifuged at 13,000 g for 10 min, and the supernatants were injected into a LC-MS/MS system for analysis.

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 LC-MS/MS system for analysis.

Determination of pyrrole-GSH conjugates The determination of pyrrole-GSH conjugates was conducted, according to a published procedure.24

An aliquot of 80 µL acetonitrile containing 12.5 ng

S-hexylglutathione 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 µL) into the LC-MS/MS for analysis.

Determination of pyrrole-protein adduction Liver pyrrole-protein 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 4-dimethylaminobenzaldehyde (v/v 4:1) in ethanol containing 1% perchloric acid at 55 °C for 10 min (Scheme 3). The resultant mixtures were mixed with 10 µL IS solution (diazepam, 1.25 µg/mL) and injected into the LC-MS/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 (5-2,000 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 ice-cold acetonitrile after 20 min incubation at 37 °C. 10 min.

The resulting mixtures were vortex-mixed and centrifuged at 19,000 g for The supernatants were dissolved with 3 volumes of water and then

analyzed by the LC-MS/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 multiple-reaction 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 UG-C18 column (250 × 4.6 mm, 5.0 µm) (Shiseido, Tokyo, Japan) protected by a Security-Guard (4.0 × 3.0 mm, 5.0 µm) C18 column (Phenomenex, Torrance, CA) which were also used for the analyses of pyrrole-GSH conjugates and pyrrole-protein

adduction.

Mobile

acetonitrile:water:formic acid (v/v/v=15:85:0.1).

phase

was

composed

of

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 µm, 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: 0-1 min, 90-90% B; 1-6 min, 90-10% B; 6-9 min, 10-90% B; 9-10 min, 90-90% 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 GSH-mBrB and m/z 392.2→246.3 (86, 24, 5) for S-hexylglutathione (IS), respectively. For determination of pyrrole-GSH 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: 0-2 min, 90-90 % B; 2-10 min, 90-10 % B; 10-12 min, 10-10 % B; 12-14 min, 10-90 % B; and 14-17 min, 90-90 % B. 0.8 mL/min.

Flow rate was

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 S-hexylglutathione (IS), respectively. For analysis of pyrrole-protein 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: 0-2 min, 70-70 % B; 2-10 min, 70-10 % B; 10-12 min, 10-10 % B; 12-14 min, 10-70 % B; and 14-17 min, 70-70 % B. rate was set at 0.8 mL/min.

Flow

The characteristic ion pairs (DP, CE, and CXP) were

m/z 341.0→252.1 (100, 40, 10) for pyrrole-protein adduction and m/z 285.1→193.5 (101, 46, 10) for diazepam (IS), respectively.



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RESULTS Hepatotoxicity of RTS and MCT Both RTS and MCT caused elevations of serum AST and ALT activities in a time-dependent 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 RTS-treated

mice were 568.0±105.1 (U/L) and 310.3±44.7 (U/L) respectively, over 3-fold higher than that of MCT-treated animals (p

Comparative Study of Hepatotoxicity of Pyrrolizidine Alkaloids

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