Role of Metabolic Activation in 8-Epidiosbulbin E Acetate-Induced

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Role of metabolic activation in 8-epidiosbulbin E acetate-induced liver injury: Mechanism of action of the hepatotoxic furanoid Dongju Lin, Weiwei Li, Ying Peng, Chunfeng Jiang, Youjun Xu, Hui-yuan Gao, and Jiang Zheng Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.5b00501 • Publication Date (Web): 17 Feb 2016 Downloaded from http://pubs.acs.org on February 27, 2016

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Chemical Research in Toxicology

Role of metabolic activation in 8-epidiosbulbin E acetate-induced liver injury: mechanism of action of the hepatotoxic furanoid †

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Dongju Lin, Weiwei Li, Ying Peng, Chunfeng Jiang, Youjun Xu, §

‡¶

Huiyuan Gao, * and Jiang Zheng * †

§

‡

School of Pharmacy, School of Traditional Chinese Medicine, Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang, Liaoning, 110016, P. R. China ¶

Center for Developmental Therapeutics, Seattle Children’s Research Institute, Division of Gastroenterology and Hepatology, Department of Pediatrics, University of Washington School of Medicine, Seattle, WA 98101

Running title: The hepatotoxicity of 8-epidiosbulbin E acetate Corresponding authors and address where work was done Jiang Zheng Center for Developmental Therapeutics, Seattle Children’s Research Institute, Division of Gastroenterology and Hepatology, Department of Pediatrics, University of Washington School of Medicine, Seattle, WA 98102, USA; Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang, Liaoning, 110016, P. R. China. E-mail:[email protected] Tel: (206) 986-7651 Fax: (206) 987-7660 Huiyuan Gao School of Traditional Chinese Medicine, Shenyang Pharmaceutical University, Shenyang, Liaoning, 110016, P. R. China Email: [email protected] Tel: +86-24-23986460; +86-24-2398648 TOC Graphic

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Abstract 8-Epidiosbulbin E acetate (EEA), a furanoid, was unexpectedly found as the most abundant diterpenoid lactone in certain Dioscorea bulbifera L. (DB), a traditional herbal medicine widely used in Asian nations. in humans and experimental animals. the commercial source.

The herb has been reported to cause liver injury The occurrence of EEA in DB was dependent on

The present study showed that EEA exhibited time- and

dose-dependent liver injury in mice.

Pretreatment with ketoconazole (KTC) prevented

the animals from EEA-induced liver injury, caused 7- and 13-fold increases in plasma Cmax and AUC of EEA, and decreased urinary excretion of glutathione (GSH) conjugates derived from EEA.

Pretreatment with buthionine sulfoximine (BSO) exacerbated

EEA-induced hepatotoxicity.

In order to define the role of furan moiety of EEA in

EEA-induced hepatotoxicity, we synthesized the tetrahydro-EEA by catalytic hydrogenation of the furan moiety of EEA.

No liver injury was observed in the animals

given the same doses of tetrahydro-EEA.

The results indicate that EEA itself does not

appear to be hepatotoxic, but the electrophilic intermediate generated by the metabolic activation of the furan ring mediated by cytochromes P450 was responsible for EEA-induced liver injury.



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Introduction Dioscorea bulbifera L. (DB), a traditional medicinal herb, has been widely used in Asian nations with a variety of pharmacological activities, including anti-tumor,1-4 antifeedant,5

anti-inflammatory,6,7

anti-viral,8

and

goiter

inhibitory

effects.9

Additionally, DB and related remedies have been clinically used to treat struma and many types of tumors for several decades.10,11 Although DB has been reported to have a wide array of therapeutic values, the adverse effects of DB also draw the attention for the safe use of the medicinal herb. Numbers of clinical case reports displayed an association of liver injury with consumption of DB and its remedies.12-15 Many diterpenoid lactones have been isolated and identified from DB, and diosbulbin B (DIOB) was reportedly the major diterpenoid found in the herb.8,16

DIOB has been reported to play an important

role in the pharmacological activity and hepatotoxicity of DB.17-19 Recently, we unexpectedly found 8-epidiosbulbin E acetate (EEA, Scheme 1) as the most abundant diterpenoid lactone in certain DB and that the occurrence of EEA in DB was dependent on the commercial source.

Our preliminary study also manifested that EEA was

hepatotoxic and more toxic than DIOB.20 Unfortunately, EEA-containing DB and EEA-free DB can both be reached on the market.

It is difficult to distinguish one from

another and there is no regulation for quality control of the consumption of DB.

Clearly,

the lack of quality control of DB places consumers to a high risk of liver injury. mechanisms of EEA-induced hepatotoxicity remain unknown.

The

Our early study

demonstrated that EEA was metabolized to a cis-enedial intermediate (2, Scheme 2), and


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the presence of ketoconazole (KTC), a P450 3A inhibitor, suppressed the production of the cis-enedial in microsomal incubations with EEA.20

cis-Enedials are known

electrophilic species that are chemically reactive to nucleophiles.21-24

As a follow-up,

the objective of the present study was to investigate the role of the metabolic activation of EEA in the liver toxicity induced by EEA.



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Materials and methods Reagents Dry rhizomes of Dioscorea bulbifera L. (DB) were obtained from Tong-Ren-Tang and Guo-Da pharmacies (Shenyang, China).

8-Epidiosbulbin E acetate (EEA) was

isolated and purified from DB rhizomes in our laboratory, according to the published procedure.25

The structure was confirmed by mass spectrometry and NMR.

The

purity of EEA was >98% determined by high-performance liquid chromatography (HPLC) with diode array detector (DAD).

Glutathione (GSH), S-hexylglutathione,

ketoconazole (KTC, >99%), and L-buthionine sulfoximine (BSO, >99%) were purchased from Sigma-Aldrich (St. Louis, MO). (Springfield, NJ).

All organic solvents were from Fisher Scientific

All reagents and solvents were of either analytical or HPLC grade.

Preparation of DB Extracts DB purchased from the two pharmacy stores were individually chopped, dried in the shade, and pulverized.

The resulting powders (30 g) were accurately weighed, soaked

in 240 mL of water for 1 h, and then boiled for 25 min as the way most traditional Chinese medicinal herbs are prepared, followed by filtration. was repeated in 150 mL of water. up to the volume of 120 mL.

The extraction process

The two batches of extracts were pooled and brought

The contents of DIOB and EEA in the two DB extracts

were determined by LC-MS/MS. Then, the extracts were concentrated in water bath at 80 °C to dryness, and reconstituted with 0.5% sodium carboxymethyl cellulose (CMC-Na) solution to 10 mL.


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Synthesis of tetrahydro-EEA Tetrahydro-EEA (1’, Scheme 1) was synthesized by selective hydrogenation of the furan moiety of EEA into tetrahydrofuran at high temperature and pressure (130 °C, 5.0 MPa) by use of Pd supported on carbon.26-28 EEA (100 mg, 0.257 mmol) was dispersed in a reactor filled with 3.0 mL tetrahydrofuran, followed by addition of 0.01 g 10% Pd/C. The reactor was tightly sealed, purged with hydrogen, pressurized to 5.0 MPa, and heated at 130 °C.

After 8 h reaction, the mixture was cooled to room temperature.

The

reaction mixture was filtered, and the filtrates were concentrated to dryness under vacuum.

The concentrates were submitted to a semi-preparative HPLC system for

product purification.

Colorless crystals were obtained with >98 % purity.

1

H NMR

(DMSO-d6, 600 MHz): δ 1.03 (3H, s, H-19), 1.43 (2H, m, H-1), 1.48 (2H, d, J = 12.9 Hz, H-10), 1.75 (3H, s, H-20), 1.78 (1H, m, H-11a), 1.80 (1H, br s, H-14a), 1.87 (1H, dd, J = 6.1, 2.9 Hz, H-7a), 1.89 (1H, d, J = 4.8 Hz, H-14b), 1.96 (1H, m, H-11b), 2.05 (1H, d, J = 12.4 Hz, H-10), 2.17 (1H, td, J = 12.0, 5.8 Hz, H-7b), 2.27 (1H, br s, H-13), 2.35 (1H, d, J = 5.8 Hz, H-5), 2.37 (1H, d, J = 7.2 Hz, H-4), 2.43 (1H, br s, H-8), 3.45 (1H, m, H-16a), 3.63 (1H, dd, J = 15.5, 7.6 Hz, H-16b), 3.73 (1H, td, J = 8.2, 4.3 Hz, H-15), 4.27 (1H, m, H-12), 4.81 (1H, m, H-6), 4.90 (1H, br s, H-2); 13C NMR (DMSO-d6, 600 MHz) δ 21.48 (C-19), 26.50 (C-1), 27.72 (C-20), 28.13 (C-14), 27.93 (C-7), 31.58 (C-10), 34.20 (C-3), 37.13 (C-11), 38.12 (C-5), 42.32 (C-4), 44.79 (C-13), 44.79 (C-8), 45.33 (C-9), 67.67 (C-15), 67.78 (C-16), 69.65 (C-12), 70.51 (C-6), 77.38 (C-2), 169.96 (C-17), 171.80



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(C-18), 177.09 (C-21); ESI-TOF MS (m/z): [MNa]+ calculated for C21H28O7Na, 415.1727; Found, 415.1721.

Animals and treatment Male Kun-Ming mice (17-20 g) were purchased from the Animal Center of Shenyang Pharmaceutical University (Shenyang, China).

Animal maintenance and

treatment were conducted in accordance with the protocols approved by the Ethics Review Committee for Animal Experimentation of Shenyang Pharmaceutical University (Shenyang, China).

Mice had free access to food and water and were housed in a

temperature-controlled (22 ± 4 ºC) facility with a 12 h dark/light cycle for at least 5 days before treatment.

EEA and tetrahydro-EEA (100 mg) was individually suspended in 10

mL of corn oil.

The animals were randomly divided into six groups, and each group

contained four mice.

One group was treated intraperitoneally (i.p.) with corn oil as the

control, and the other groups were given EEA at dosages of 50, 75, 100, or 150 mg/kg, or tetrahydro-EEA (100 mg/kg), respectively.

The animals were sacrificed 12, 24, 36, and

48 h after the treatment. In a separate study, mice were pretreated with BSO as previously described.29,30 Briefly, mice were treated with BSO (666 mg/kg, i.p.) in saline. administration, the animals were given EEA at 100 mg/kg (i.p.). collected 36 h after the administration.

After 1 h of the

Blood and livers were

Another group of mice were treated (i.p.) with


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KTC (100 mg/kg).

After 1.5 h of the administration, the animals were treated with EEA

(i.p.) at 100 mg/kg and sacrificed 36 h after the administration.

Mice in a separate group were orally administered EEA-containing DB extracts (0.8 mL/20 g), EEA-free DB extracts (0.8 mL/20 g), DIOB dissolved in 0.5% CMC-Na solution (150 mg/kg) at the dose equivalent to that of DIOB existing in EEA-free extracts (0.8 mL/20 g) or a mixture of DIOB (1.8 mg/kg) and EEA (155 mg/kg) dissolved in 0.5% CMC-Na solution at the dose equivalent to that of the two test compounds found in the extracts of the EEA-containing DB (0.8 mL/20 g), respectively.

The mice were

sacrificed 36 h after the administration, and the serum ALT and AST levels were measured.

ALT and AST Assays The blood samples collected above were allowed to clot in test tubes at room temperature for 1 h, followed by centrifuging at 8,000 g. harvested for ALT and AST assays.

The resulting sera were

Serum ALT and AST activities were measured by

VITROS® 5600 Integrated System (Orthos-Clinical Diagnostics, Rochester, NY).

Histopathologic Examination The liver tissues were fixed in 10% neutral buffered formalin, paraffin processed, and sectioned at 3 µm.

For histological evaluation, the tissue sections were stained with

hematoxylin and eosin (H&E).



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Toxicokinetic study Mice were randomly divided into two groups, and each group included 40 mice and time point of the toxicokinetic study contained 4 mice.

Mice in group I were given EEA

(100 mg/kg, i.p.) alone, and mice in group II were pretreated with KTC as described above and then received EEA (100 mg/kg, i.p.) 1.5 h after the pretreatment.

Blood

samples were collected from the retro-orbital sinus at time intervals 10, 20, 30, and 45 min and 1.0, 2.0, 4.0, 6.0, 8.0, and 12.0 h after EEA injection.

Aliquots (50 µL) of

plasma prepared as described above were mixed with 150 µL of acetonitrile containing internal standard S-hexylglutathione (final concentration: 10 ng/mL).

The resulting

mixture was vortex-mixed and centrifuged at 19,000 g for 10 min to remove precipitated protein.

The resultant supernatants (5 µL) were injected onto an LC-MS/MS system for

analysis as described below.

A non-compartmental model was utilized to describe the

plasma concentration time profile of EEA.

The values of peak plasma concentrations

(Cmax), area under the plasma concentration-time profiles (AUC), time required to reach maximum plasma concentration (Tmax), apparent volume of distribution (Vz/F), and apparent clearance (CL/F) were determined for each mice using Drug And Statistic (DAS) 2.1.1 software (Mathematical Pharmacology Professional Committee of China, Shanghai, China).

Preparation of urine samples


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In a separate study, mice were placed in metabolism cages after treatment.

Urine

samples were collected from 0-24 h post procedure. Aliquots (100 µL) of urine samples collected were mixed with 300 µL acetonitrile containing internal standard S-hexylglutathione (final concentration: 10 ng/mL).

After centrifugation, the

supernatants were harvested and evaporated to dryness under a stream of nitrogen gas at 40 °C.

The resulting residues were reconstituted with 100 µL of 50% acetonitrile in

water and centrifuged at 19,000 g for 10 min, and the supernatants (5 µL) were injected onto the LC-MS/MS for analysis.

LC-MS/MS analysis All samples were analyzed on a 4000 Q-TrapTM LC-MS/MS system, a hybrid triple quadrupole/LIT (linear ion trap) mass spectrometer (Applied Biosystems, Foster City, CA) equipped with a TurboIonSpray® ion source.

The chromatographic separation was

achieved on a Thermo 5.0 µm BDS HYPERSIL C18 ODS column (150 × 4.6 mm) (Thermo, San Jose, CA).

The mobile phase consisted of acetonitrile with 0.1% formic

acid (A) and 0.1% formic acid in water (B) with a gradient elution of 20% A at 0-2 min, 20-90% A at 2-10 min, 90-90% A at 10-12 min, 90-20% A at 12-14 min, and 20-20% A at 14-15 min.

The HPLC flow rate was 0.8 mL/min.

performed on a 5 µL aliquot of samples.

LC/MS/MS analyses were

The characteristics of ion pairs (corresponding

to declustering potential, DP; collision energy, CE; collision cell exit potential, CXP) for EEA was m/z 389.2→311.6 (141, 25, 3), and those for EEA-GSH conjugates and



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S-hexylglutathione (internal standard) were m/z 676→616 (110, 50, 3), and m/z 392.2 → 246.3 (86, 24, 5), respectively.

Data were processed using Applied Biosystems/SCIEX

AnalystTM software (versions 1.6 and 1.6.1).

Statistics Drug and Statistic (DAS) 2.1.1 software (Mathematical Pharmacology Professional Committee of China, Shanghai, China) was used for profiling of the toxicokinetic data. Unless stated otherwise, data are presented as mean ± SD.

Statistical analyses were

performed by unpaired Student's t-tests or ANOVA, with Student-Newman-Keuls tests post hoc, using Graph Pad Prism software.

A value of p

Role of Metabolic Activation in 8-Epidiosbulbin E Acetate-Induced

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