Proteomic Investigation of Signatures for Geniposide-Induced

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Proteomic Investigation of Signatures for Geniposide-Induced Hepatotoxicity Junying Wei, Fangbo Zhang, Yi Zhang, Chunyu Cao, Xianyu Li, Defeng Li, Xin Liu, Hongjun Yang, and Luqi Huang J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/pr5007119 • Publication Date (Web): 22 Oct 2014 Downloaded from http://pubs.acs.org on October 26, 2014

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Journal of Proteome Research

Proteomic Investigation of Signatures for Geniposide-Induced Hepatotoxicity

Junying Wei1#, Fangbo Zhang1#, Yi Zhang1, Chunyu Cao1, Xianyu Li2, Defeng Li1, Xin Liu1, Hongjun Yang1∗, Luqi Huang3* 1 Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China； 2 State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 102206, China; 3 State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China

Abstract Evaluating the safety of traditional medicinal herbs and their major active constituents is critical for their widespread usage. Geniposide, a major active constituent with a defined structure from the traditional medicinal herb Gardenia jasminoides ELLIS fruit, exhibits remarkable anti-inflammatory, anti-apoptotic and antifibrotic properties and has been used in a variety of medical fields, of which mainly for the treatment of liver diseases. However, geniposide-induced hepatotoxicity and methods for the early detection of hepatotoxicity have yet to be reported. In this study, geniposide-induced hepatotoxicity was investigated. In addition, candidate biomarkers for the earlier detection of geniposide-induced hepatotoxicity were identified using a label-free quantitative proteomics approach on a geniposide overdose-induced liver injury in a rat model. Using an accurate intensity-based absolute quantification (iBAQ)-based one-step discovery and verification approach, a candidate

# These authors contribute equally. ∗To whom correspondence should be addressed. E-mail: [email protected] Phone: 8610-64032656. Fax: 8610-64013996 ； [email protected] Phone: 8610-84035184. Fax: 8610-64013996.

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biomarker panel was easily obtained from individual samples in response to different conditions. To determine the biomarkers’ early detection abilities, five candidate biomarkers were selected and tested using enzyme-linked immunosorbent assays (ELISAs). Two biomarkers, glycine N-methyltransferase (GNMT) and glycogen phosphorylase (PYGL), were found to indicate hepatic injuries significantly earlier than the current gold standard liver biomarker. This study provides a first insight into geniposide-induced hepatotoxicity in a rat model and describes a method for the earlier detection of this hepatotoxicity, facilitating the efficient monitoring of drug-induced hepatotoxicity. Keywords: Proteome ● Geniposide ● Hepatotoxicity ● Biomarker

INTRODUCTION Evaluating the safety of traditional medicinal herbs and their major active constituents is critical for the widespread usage of these herbs. Scientists around the world are striving to fulfill the promise of effectively identifying the onset of early toxicity with these herbs. Geniposide, an iridoid glycoside compound with a defined structure, is the major active constituent of the Gardenia jasminoides ELLIS fruit, which is commonly used as a traditional medicine herb for the treatment of liver diseases in many Asian countries 1-4. After oral ingestion, geniposide is converted to the active metabolite genipin by intestinal bacteria 5. Geniposide and genipin possess remarkable choleretic 7-11

，anti-apoptotic

1, 2, 12

，antifibrotic

13-15

and neuroprotective

16

3, 6

，anti-inflammatory

properties. In addition, genipin is a

natural crosslinking agent 17 that has a high biocompatibility, resulting in widespread use of genipin in a variety of medical fields, including nerve regeneration

18

and tendon repair

19

. Because of the

increasing applications of geniposide and genipin, a toxicity assessment of these compounds as well as a method for monitoring toxicity are urgently needed. To investigate geniposide- and genipin-induced toxicity, the genotoxicity of the components of gardenia yellow was evaluated

20

. Gardenia yellow can be extracted from the Gardenia jasminoides

ELLIS fruit with ethanol. Experimental results indicated that genipin possesses genotoxicity. Another investigation demonstrated that genipin induces apoptotic cell death in FaO rat hepatoma cells and human hepatocarcinoma Hep3B cells. In addition, observations in that study implied that genipin


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signaling of apoptosis of hepatoma cells is mediated via NADPH oxidase-dependent generation of reactive oxygen species (ROS), which leads to downstream of c-Jun NH2-terminal kinase (JNK) 21. To investigate the relationship between geniposide levels and toxicity, a 13-week oral dose subchronic toxicity study of gardenia yellow containing geniposide was conducted in rats, and a safe dose of geniposide was determined

22

. Additional studies were also performed to investigate the cytotoxic

effects of geniposide and its metabolite genipin and to determine the role of geniposide and genipin in geniposide-induced cytotoxicity. The results of these studies indicated that genipin increases cytotoxic effects in cells while geniposide does not 23. Although several investigations have been performed, the knowledge of geniposide- and genipin-induced toxicity is not comprehensive, and there are relatively few studies on the relationship between geniposide intake and hepatotoxicity. The liver is central to drug metabolism and may be involved in the biotransformation of geniposide. However, to our knowledge, neither geniposide-induced hepatotoxicity nor methods for early detection of such hepatotoxicity have been investigated. In recent years, mass spectrometry-based proteomics has been widely used to determine the modes of action and mechanisms involved in drug- or chemical-induced toxicity

24-31

. Due to the superior

comprehensiveness of proteomics and its powerful quantitative approach, the new field of toxicoproteomics offers a unique opportunity to identify signatures and biomarkers of hepatotoxicity at an early stage

32-34

. Despite the popularity of proteomics, the limited amount of information on

geniposide-induced hepatotoxicity makes the discovery, analysis, and validation of biomarkers for toxicity monitoring challenging. First, a testing model must be chosen. Both in vitro and in vivo testing can be performed. However, to deem a compound safe for human consumption, the compound should be tested in vivo on rodents, especially on rats

32, 35

. When testing on rodents, liver tissue or

cellular fractions, such as mitochondria, endoplasmic reticulum and microsomes, or blood 36, 37, can be used. However, the lack of knowledge regarding the mechanism of drug action makes liver tissue the preferred testing medium. Second, in the biomarker discovery phase, the number of rodents tested under a particular condition must be sufficient for statistical analysis, as each individual’s response to geniposide intake may differ. With the conventional approach, multiple samples under the same



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conditions are pooled and analyzed using quantitative proteomics; however, the resulting differences must still be verified against individual samples. To avoid redundant verification, proteins can be directly quantified from individual rather than pooled samples. Candidate biomarkers can then be identified by a statistical analysis of all of the protein quantifications in response to different conditions. The challenge is how to accurately quantify the proteins from multiple samples. While the most powerful label-based quantitative approach can quantify the proteins from more than 8 samples 38, 39

, the number of samples that can be analyzed in a single experiment is still limited. In contrast, a

label-free quantitative approach has no such sample limitation, and this approach’s ability to accurately quantitate many more samples makes it the best choice to address this problem. Finally, the discovered and validated signature biomarkers for geniposide-induced hepatotoxicity should show potential for clinical applications. To realize this potential, the proposed biomarkers should be carefully selected based on certain criteria. For example, the biomarkers should be detectable in blood at an appropriate concentration, have the necessary liver specificity and human orthologs. Moreover, though a search in public databases may be performed to discover some potential protein biomarkers, the actual investigations are still urgently needed to find sensitive measures to report injuries significantly earlier than the currently used liver biomarker 37. In this work, we applied a label-free quantitative proteomics approach to geniposide overdose-induced liver injury in rat models to investigate the action of geniposide-induced hepatotoxicity and to identify candidate biomarkers for earlier monitoring. Using an accurate intensity-based absolute quantification (iBAQ) 40-based protein quantification approach, the amounts of proteins from individual samples in response to different conditions were measured. Candidate biomarkers were then identified using a one-step discovery and verification approach and statistical analyses of the measured protein amounts. Finally, to facilitate earlier monitoring of geniposide-induced hepatotoxicity, five candidate biomarkers were selected and confirmed by enzyme-linked immunosorbent assays (ELISAs). This study provides a first insight into geniposide-induced hepatotoxicity in a rat model and describes an efficient method for earlier hepatotoxicity detection. EXPERIMENTAL SECTION


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Materials and Reagents Geniposide with over 98% purity (by HPLC) was obtained from Shanyun Biochemical Science and Technology Co., Ltd (Guangxi, China). Sequencing grade porcine trypsin and dithiotheitol (DTT) were obtained from Promega (Madison, WI, USA). Iodoacetamide (IAA) and formic acid (FA) were purchased from Acros (Morris plains, NJ, USA). Protease inhibitor cocktail tablets were obtained from Roche (Mannheim, Germany). HPLC-grade acetonitrile (ACN) was obtained from Mallinckrodt Baker Inc (Phillipsburg, NJ, USA). All other chemicals were of analytical grade reagent. Deionized water (R>18.2 MΩ) used for all experiments was purified by using Millipore purification system (Billerica, MA, USA). Animal Study After 24 hours of fasting, male Sprague–Dawley (SD) rats (Vital River Laboratories, Beijing, China), nine weeks of age, were administered high-dosage geniposide (300 mg/kg daily), medium-dosage geniposide (100 mg/kg daily), low-dosage geniposide (30 mg/kg daily) or saline (control) by oral administration (p.o.) for three consecutive days. Both geniposide-treated animals and control animals were euthanized one, two, and three days post-intake. Plasma samples were collected, and after washed out blood by perfusion with saline, liver tissues were harvested. Tissue samples were frozen at -80°C for proteomic analysis. Plasma samples were collected in tubes and frozen at -20°C for analysis. Alanine transaminase (ALT) and aspartate transaminase (AST) levels in sera were determined by an enzymatic assay (WanTai DRD, Beijing, China). All animal experiments were approved by the Committee on Animal Care and Use of Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences. Histopathology The liver samples were stained with hematoxylin and eosin (H&E). After fixation, the liver was embedded in paraffin and sectioned at 5 µm intervals. The tissue sections were repeatedly washed with decreasing concentrations of ethanol prior to eosin staining. The liver was then mounted, and its morphology was examined by pathologists and imaged via microscopy (DMI6000 B microscope, Leica, Wetzlar, Germany).



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Preparation of Protein Samples Rat livers were homogenized in the PBS (KCl: 0.2 g, KH2PO4: 0.2 g, NaCl: 8.0 g, Na2HPO4·12H2O: 3.9054 g, pH 7.4, 1000 mL) buffer containing cocktail

41

with a high-throughput tissue homogenizer

(Sceintz-48, Sceintz, Ningbo, China). Then proteins of rat livers were extracted with 8M urea, and 300 µg (44.79 µL) of protein was reduced by adding 4.98 µL of 0.1 M DTT for 4 h at 37 °C and then alkylated by adding 5.53 µL of 0.5 M iodoacetamide for 60 min at room temperature in the dark. The protein sample was finally digested using trypsin in 50 mM ammonium bicarbonate (pH 8.0) at a mass ratio of 1:50 enzyme/protein for 24 h at 37 °C. LC-ESI-MS/MS Measurement Peptides were dissolved with 0.1% FA in water solution, and then analyzed on 2-D nanoLC (Eksigent, USA) coupled to 5600 Triple-TOF (Applied Biosystems) by an in house made C18 column (75 µm inner-diameter, 360 µm outer-diameter × 10 cm, 3 µm C18) with a flow rate of 350 nL/min. Mobile phase A consisted of 0.1% FA in water solution, and mobile phase B consisted of 0.1% FA in acetonitrile solution. The solvent gradient was set as follows: 5%-8% B, 5 min; 8%-18% B, 35 min; 18%-32% B, 22 min; 32%-95% B, 2 min; 95% B, 4 min; 95%-5% B, 4 min. The MS conditions were as the followings 42, 43: Nano-spray ion source was used. A spray voltage of 2600 V was applied. The MS scan range was m/z 350–1250. The top 50 precursor ions were selected in each MS scan for subsequent MS/MS scans. MS scans were performed for 0.25 s, and subsequently 50 MS/MS scans were performed for 0.04 s of each. The dynamic exclusion for MS/MS was set as 12 s. The CID energy was automatically adjusted by the rolling CID function of Analyst TF 1.5.1. Protein Identifications and Quantifications Wiff files from 5600 Triple-TOF were searched by ProteinPilot version 4.2 using Paragon search engine against the rat ref-sequence protein database (updated on 03-31-2014). The false positive rate was set at 1% on the peptide level. Mascot generic format (Mgf) files containing MS peak lists were exported by ProteinPilot, and then iBAQ-based protein quantifications

40, 42

were performed by an

in-house software. Briefly, the iBAQ intensities were obtained by dividing the protein intensities by the number of theoretically peptides which were calculated by in silico protein digestion with a PERL


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script, and all fully tryptic peptides between 6 and 30 amino acids were counted while missed cleavages were neglected. ELISA Samples of whole blood harvested from animals were centrifuged at 10,000 rpm for 10 min at 4 °C. The resultant serum was then assayed using ELISA kits from CUSABIO life science Co. Ltd. (Wuhan, China) according to the manufacturer’s instructions. Five ELISA kits, rat glycine N-methyltransferase (GNMT) ELISA kit, rat glycogen phosphorylase (PYGL) ELISA kit, rat α-enolase ELISA kit, rat alanine--glyoxylate aminotransferase 2 (AGXT2) ELISA kit and rat aldehyde dehydrogenase 1 L1(ALDH1L1) ELISA kit were used. Each kit consists of a 96-well plate into which a specific antibody against a target protein is immobilized. The target protein in sera is recognized by the antibody, followed by incubation with a horseradish peroxidase-conjugated secondary antibody for the colorimetric quantification. The plates were read on a microplate reader (Molecular Devices, USA) at 450 nm. The reactions were carried out in triplicate for each sample. Finally, the results were analyzed by one-way analysis of variance, and significant at P

Proteomic Investigation of Signatures for Geniposide-Induced

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