Global and Targeted Metabolomics Reveal That Bupleurotoxin, a

Dec 16, 2013 - School of Pharmacy, Second Military Medical University, Shanghai 200433, PR ... School of Chinese Medicine, Hong Kong Baptist Universit...
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Global and Targeted Metabolomics Reveal That Bupleurotoxin, a Toxic Type of Polyacetylene, Induces Cerebral Lesion by Inhibiting GABA Receptor in Mice Zhongxiao Zhang,†,# Cheng Lu,§,# Xinru Liu,†,# Juan Su,† Weixing Dai,† Shikai Yan,‡ Aiping Lu,§,∥,* and Weidong Zhang†,‡,* †

School of Pharmacy, Second Military Medical University, Shanghai 200433, PR China School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, PR China § Institute of Basic Research in Clinical Medicine, China Academy of Chinese Medical Science, Beijing 100700, PR China ∥ School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong ‡

S Supporting Information *

ABSTRACT: Polyacetylenes are widely distributed in food plants and medicinal herbs, which have been shown to have highly neurotoxic effects. However, there were insufficient studies on the toxicity of these compounds. Thus, a series of experiments was designed to elucidate the toxicity mechanism of bupleurotoxin (BETX) as a representative polyacetylene. First, male BALB/c mice were intragastrically administered 2.5 mg/kg of bodyweight BETX once a day for seven consecutive days. The histopathological results showed that BETX could induce severe morphological damages in the brain hippocampus. We then used metabolomics approaches to screen serum samples from the control and BETX-treated groups. The global metabolomics results revealed 17 metabolites that were perturbed after BETX treatment. Four of these metabolites were then verified by targeted metabolomics. Bioinformatics analysis with the Ingenuity Pathway Analysis (IPA) software found a strong correlation between the GABA receptor signaling pathway and these metabolites. On the basis of these results, a validation test using a rat hippocampal neuron cell line was performed, and the results confirmed that BETX inhibited GABA-induced currents (IGABA) in a competitive manner. In summary, our study illustrated the molecular mechanism of the toxicity of polyacetylenes. In addition, our study was instructive for the study of other toxic medical herbs. KEYWORDS: polyacetylenes, bupleurotoxin, metabolic profiles, neurotoxicity, GABA receptor

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INTRODUCTION It is known that polyacetylenes are widely distributed and are found in food plants, coating of the tongue, sponge, fungus, seaweed, lichen, tunicate, hexapod, frogs, and in trace amounts in humans.1 Polyacetylenes have been indicated to be highly toxic to bacteria, fungi,2 and mammal cells, to show neurotoxic activity,3−5 anti-platelet-aggregatory effects,6−8 and anti-inflammatory,9,10 and to induce allergic skin reactions. These compounds were also found to have the effects on human cancer cells, which could inhibit tumor formation in vivo model.11,12 All of these human bioavailabilities indicated that polyacetylenes might be beneficial to human health.13 Unfortunately, the toxicity of these compounds has not been sufficiently studied. To reveal the mechanisms responsible for the toxic effects of these compounds, we selected bupleurotoxin (BETX) isolated from Bupleurum longiradiatum as a neurotoxicity-inducing compound. In the medical history, B. © 2013 American Chemical Society

longiradiatum has been misused as a substitute for the traditional Chinese medicinal herb Bupleuri Radix and has caused several cases of human poisoning, some of which have resulted in death.14 In the previous study, we found that polyacetylenes isolated from the roots of B. longiradiatum were responsible for the main toxicity of this herb.15,16 Given the toxicity of these compounds and their broad distribution in the medical herb, we had developed a high-performance liquid chromatography method coupled with a diode array detector and mass spectrometry (HPLC−DAD−MS) platform for the qualitative and quantitative analysis of these compounds.14 However, we also need to clarify the mechanism responsible for the toxic effects of these compounds and provide guidance for their safe use. Received: September 25, 2013 Published: December 16, 2013 925

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confirmed according to a preliminary experiment. After the last administration, blood was collected, and all of the mice were sacrificed. Fresh brain, liver, heart, and kidney were obtained and fixed in cold (4 °C) 10% neutral buffered formalin before paraffin-embedded. These organs (liver, heart, and kidney) were sectioned at 5 μm and stained with hematoxylin and eosin (H&E) for histological examination. Sections of the brain (8-μm thick) were stained with TUNEL assay using the In Situ Cell Death Detection Kit (Roche catalog no. 11684817910). First, the brain was fixed in 4% paraformaldehyde (PFA) and sectioned; second, the brain sections were preincubated in PBST (0.1 M PBS plus 1% Triton) and then labeled by the In Situ Cell Death Detection Kit for 1 h at 37 °C. At last, the sections of brain were washed in PBST and observed by the electron microscope. The whole blood was centrifuged at 3500 rpm for 15 min at 4 °C to obtain serum. Then serum was transferred into glass tube, which was stored at −80 °C for further analysis. All experiments in this study conformed to the guidelines for animal research. All of animals were handled humanely throughout the experiment.

A blood sample is an accessible biofluid for the discovery of potential biomarkers of toxicity. The blood metabolites are obvious potential biomarkers since they are the end products of enzymatic reactions and other bioconversions and are smaller than the number of proteins.17 Metabolomics, an emerging -omics approach corresponding to metabolites, has been used to evaluate biological effects of tissue injury and/or the toxicity mechanisms of toxins and drugs.18−20 Metabolomics can provide an instantaneous snapshot of metabolic alterations, which in turn provides a view of the whole metabolism process and thereby crucial information on the toxicity through the identification of the changed metabolites and the relevant metabolic pathways by measuring many endogenous small molecules. In the current study, liquid chromatography time-of-flight mass spectrometry (LC−Q-TOF-MS)-based global metabolomics and targeted metabolomics were used to screen the perturbations in the metabolite profile of mice after BETX treatment. And the biological networks were constructed using the IPA software. In addition, we validated the hypothesis using a rat hippocampal neuron cell line.

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Global Metabolomics Analysis

The LC−Q-TOF-MS analysis was performed on an Agilent1200 LC system coupled with an electrospray ionization (ESI) source and an Agilent-6520 Q-TOF mass spectrometer (Agilent Technologies, CA, USA) in both positive and negative modes with MassHunter acquisition software (version B.02.00), as described in the previous literature.21 However, the data of positive mode was utilized for further research, since the metabolites were detected with greater ion intensities in the positive mode. And total ion chromatographies (TICs) were more stable in the positive mode. Besides, no metabolites were detected only in the negative mode.22 Briefly, a 100 μL aliquot of serum was added with 300 μL of methanol and vortex mixed for 30 s. And then the mixture was centrifuged at 12000 rpm for 10 min at 4 °C; 200 μL aliquot of water was added with 200 μL of the supernatant, vortex mixed well, and then stored at −80 °C until analysis.23 An Eclipse plus C18 column (1.8 μm, 3.6 mm × 100 mm, Agilent) was applied for the LC analysis of all samples. The column temperature was maintained at 45 °C and eluted with a 2−100% acetonitrile−ultrapure water (0.1% (v/v) formic acid) at the flow rate 250 μL/min for 19 min. A 3 μL aliquot sample was injected into volume. The gradient duration program was used as follows: 0−2 min, 2% acetonitrile; 2−3 min, 2−20% acetonitrile; 3−11 min, 20− 70% acetonitrile; 11−12 min, 70−95% acetonitrile; 12−19 min, 100% acetonitrile, and re-equilibrated for 3 min. The parameters used for the mass detection were the following: drying gas nitrogen (N2), 8 L/h; gas desolvation temperature, 330 °C; pressure of nebulizer, 35 psig; capillary voltage, 3900 V. The MS/MS data were acquired in the targeted MS/MS mode with collision energy of 10, 20, and 40 eV to identify potential biomarkers. During metabolomic profiling, the data were acquired in the continuous mode and averaged over 10 scans. The full scan mass range was 80−1000 m/z.

MATERIALS AND METHODS

Chemicals and Reagents

Dichloromethane (CH2Cl2), chloroform (CHCl3), ethanol, hexane, ethyl acetate (EtOAc), and methanol (analytical-grade) were all obtained from Shanghai Chemical Reagent Company (Shanghai, China). Methanol and acetonitrile (MS grade) were both obtained from Honeywell Burdick and Jackson (Muskegon, MI, USA). Formic acid (mass spectroscopic grade) was obtained from Fluka (Buchs, Switzerland). Ultrapure water was produced through a Milli-Q system (Bedford, MA, USA). Dimethyl sulfoxide (DMSO) and other chemical standards were obtained from Sigma-Aldrich (St. Louis, MO, USA). Extraction and Identification of BETX

The plant of B. longiradiatum collected from Shangzhi Town, Heilongjiang Province, People’s Republic of China was identified by Prof. Hanming Zhang, Department of Pharmacognosy, Second Military Medical University. The air-dried roots of B. longiradiatum (2.0 kg) were extracted as previously described.15 The purity of BETX was more than 98%. The compound was identified as bupleurotoxin based on unequivocal assignments of its NMR spectroscopic data and through comparing with other data, including UV spectrum, IR spectrum, and optical rotation.15 Animal Treatment and Sample Preparation

Male BALB/c mice (20 ± 2 g) obtained from the Slac Laboratory Animal Co., Ltd. (Shanghai, China) were housed individually in stainless-steel metabolic cages and allowed free access to standard food and filtered tap water with a 12-h light/ 12-h dark cycle. The house temperature was maintained at 25 ± 2 °C, and the relative humidity was maintained at 50 ± 10%. After acclimatization, the mice were randomly divided into two groups: control group (10 mice) and BETX-treated group (20 mice). BETX was dissolved in DMSO and then diluted with physiological saline up to the appropriate concentration (concentration of DMSO was less than 0.1%). The mice in the BETX-treated group received an i.p. administration of 2.5 mg/kg bodyweight of BETX once a day for seven consecutive days, and the mice in the control group were administered an equivalent volume of saline. The dosage of BETX was

Data Processing and Metabolite Identification

The raw LC−Q-TOF-MS data were first processed using the Agilent Mass Hunter Qualitative Analysis and Mass Profiler software (Agilent Technologies, CA, USA). Centroid LC−MS data sets from positive mode were processed with the “find compounds by molecular feature” in the Mass Hunter Qualitative Analysis Software (version B.02.00, Agilent Technologies). The parameters were optimized to improve 926

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15 V). 5 μL of each sample was injected into the column with a 19 min gradient dilution from 2 to 100% of acetonitrile, and the column and mobile phases were same as the global metabolomics study. The peaks were integrated by the instrument software. The relationships between these verified metabolites were investigated through a network construction by the IPA software.

the extraction of metabolite information, and the parameters were chosen as follows: the range of m/z values was from 80 to 1000, and peak filters were set to centroid height with more than 100 counts, and compound filters set the base peak to more than 1000 counts. This processing step generated MHD files (containing retention times and neutral mass), and further processing was performed with Mass Profiler software (version B.02.00, Agilent Technologies), which was used to align mass features across multiple LC−MS data files. The tolerances of retention time shift and the mass accuracy were set at 0.1 min and 30 ppm, and the data were acquired from 0 to 19 min. 5170 ions (mass-retention time pairs) detected in at least 80% of samples of each group were utilized for further analysis.24 The data set of these 5170 mass features was then normalized using the sum intensity. The multivariate data matrix was analyzed using the SIMCA-P software (version 11, Umetrics, Umea, Sweden), and the data were pareto scaled.25 Then an orthogonal partial least-squares analysis (OPLS) was used to find those ions that significantly contributed to the distinction of the samples and to exclude uncorrelated variables. The statistical significance was analyzed by one-way analyses of variance (ANOVAs) and Duncan’s posthoc tests by SPSS 13.0 version software (SPSS Inc., Chicago, IL, USA). Confidence level was set at 95% (P < 0.05) to determine the significance of difference. Metabolite identification was carried out by searching accurate masses of compounds in the HMDB (www.hmdb.ca) database, METLIN (www.metlin.scripps. edu), and KEGG (www.genome.jp/kegg). In addition, confirmation with available reference standards was performed by comparing their accurate mass, retention time, and MS/MS fragments with those metabolites detected in serum. All serum samples were randomized during sequence analysis. And the pooled quality-control (QC) sample was injected at the beginning of the sequence and then was injected every six serum samples throughout the whole experiment to evaluate the stability of the system (n = 36, including six QCs); the sequence was analyzed within one day per mode.

Validation Test Based on Cell Line

On the basis of the specific cerebral lesion and GABA receptor signaling pathway, which was represented clearly by the bioinformatics analysis of the metabolite profiles obtained, a validation cell line assay was conducted to clarify whether BETX impede GABA binding to the gamma-aminobutyric acid type A receptors (GABAARs) on cultured rat hippocampal neurons by the whole-cell patch-clamp technique. The rat primary hippocampal neurons were cultured according to a previously reported protocol.28 During the electrophysiological recording, the neurons were bathed in the standard external solution unless where otherwise mentioned. BETX was applied using a well-established method named “Y-tube”.29 The tip of Y-tube was placed at a site 50−100 μm from the neuron patch. The GABA-induced currents termed IGABA in the presence or absence of BETX were recorded. The patch clamp recordings were carried out under voltage clamp conditions for the conventional whole-cell current analysis. Patch pipettes were made by pulling the glass capillaries with an outer diameter of 1.5 mm using a two-stage puller (Model PP-830) that was purchased from Narishige Co., Ltd. The patch pipet had an open tip resistance of 3−5 MΩ under the present external and internal solutions. A Multiclamp 700A device (Molecular Devices) was used to record the membrane currents. A Digidata 1320A interface and a computer running the Clampex and Clamp-fit (version 10.0.1, Axon Instruments) software programs were used to sample and analyze the electrophysiological data, respectively.

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Molecular Pathway and Network Analysis in IPA

RESULTS

Identification of BETX

The Ingenuity Pathway Analysis (IPA) software (Ingenuity, Redwood City, CA, USA) is web-based software that provides comprehensive bioinformatics analysis about the interplays among metabolites, proteins, gene pathways, cell phenotypes, and disease progression.26,27 In our study, the network construction was performed by uploading metabolites IDs (HMDB number) and quantitative data into IPA to discover the biological relationships between these metabolites.

BETX was identified as bupleurotoxin on the basis of its NMR data (shown in Figure S1, Supporting Information) and through comparing with the data in previous literature (including UV spectrum, IR spectrum, and optical rotation data).15 The structure was shown in Figure 1.

Targeted Metabolomics of Identified Metabolites

On the basis of the histopathological results and global metabolic profiles, the metabolomic response of the nervous system obtained through a literature search was re-examined in a new targeted metabolomics analysis. In this experiment, a new set of animals was used, and the animal treatment and serum sample collection were the same as those used for the identification of the global metabolic profiles. The methods of serum extraction were consistent with those used for the global metabolomics study. The detection was conducted using an Agilent 6410 triple-quadrupole mass spectrometer (Palo Alto, CA, USA) with the multiple reaction monitoring (MRM) transitions for kynurenic acid (precursor → 144; collision energy, 16 V), kynurenine (precursor → 94; collision energy, 8 V), 3-hydroxykynurenine (precursor → 110; collision energy, 12 V), and quinolinic acid (precursor → 150; collision energy,

Figure 1. Structure of BETX.

Histological Characterization

There were no histopathological abnormalities in the heart, kidney, and liver after BETX treatment (shown in Figure S2, Supporting Information). In contrast, two kinds of cells were found in the brain samples. The nucleus of the type I cells (control group) in the hippocampus of the brain exhibited the morphological appearance of normal cells, which lacked nuclear condensation and fragmentation (Figure 2A,C), which indicates their neuronal nature. The apoptotic cells (BETX-treated 927

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Figure 2. TUNEL staining of the brain hippocampus. (A,C) Control group. The shape and size of the nuclear profiles clearly indicate the neuronal nature of the cells. (C,D) BETX-treated group. The nucleus of this cell in has the morphological features of apoptosis, as characterized by the dark staining. TUNEL staining. Magnification: 200× (A,B); 400× (C,D).

Figure 3. Results of the multiple pattern recognition of serum metabolites in the control and BETX-treated groups. (A) OPLS score plot (R2Y = 0.98, R2X = 0.56, and Q2 = 0.97); left (red ▲), control; right (black ■), BETX-treated group. (B) OPLS S-plot. Each triangle in the S-plot represents an ion. The ions far away from the origin represent potential biomarkers.

principal components analysis (PCA) showed that the QC samples were tightly clustered (shown in Figure S3, Supporting Information). Moreover, the retention times, mass accuracies, and peak areas of the five selected EICs in the QC samples also showed good system stability. The RSDs of the mass accuracies, retention times, and peak areas of the five peaks were 0.12E− 04%−0.70E−04%, 0.03−0.92%, and 4.57−14.34%. These results demonstrated that the reliability and stability were qualified for the whole sequence in this study.

group) in the hippocampus of the brain were dark stained with fragmented cell nuclear and condensed chromatin (Figure 2B,D). Sample Repeatability and LC−MS System Stability

To assess the repeatability of the method used in this study, extracts from six aliquots of a random serum sample were continuously injected. And we selected five common extracted ion chromatograms (EICs) with different m/z values and polarities, which were shared by these injections. The relative standard derivations (RSDs) of the peak areas and retention times of these peaks were 4.52−13.31% and 0.05−0.90%, respectively. The stability of the LC−MS system for sequence analysis was evaluated by testing the pooled QC samples. The

Identification of Significant Metabolites through Global Metabolomics

The total ion chromatograms (TICs) of serum were acquired from both control and the BETX-treated groups. To maximize 928

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Table 1. Metabolites Identified in the Control and BETX-Treated Groupsa exact mass

name

formula

ID

trendb

P-value

fold change

174.1117 226.1066 240.1222 115.0633 224.0797 297.1073 230.0192 252.0746 208.0848 236.0797 167.1189 138.0429 152.0473 189.0426 322.2508 318.2195 304.2402

arginine carnosine homocarnosine proline 3-hydroxykynurenine methylguanosine D-ribose 5-phosphate 5-hydroxy-N-formylkynurenine kynurenine formylkynurenine quinolinic acid urocanic acid 3-hydroxyphenylacetic acid kynurenic acid 15(s)-HETrE 14,15-EpETE arachidonic acid

C6H14N4O2 C9H14N4O3 C10H16N4O3 C5H9NO2 C10H12N2O4 C11H15N5O5 C5H11O8P C11H12N2O5 C10H12N2O3 C11H12N2O4 C7H5NO4 C6H6N2O2 C8H8O3 C10H7NO3 C20H34O3 C20H30O3 C20H32O2

HMDB03416 HMDB00033 HMDB00745 HMDB00162 HMDB11631 HMDB01563 HMDB00618 HMDB04086 HMDB00684 HMDB60485 HMDB00232 HMDB00301 HMDB00440 HMDB00715 HMDB05045 HMDB10205 HMDB01043

↑ ↑ ↑ ↑ ↑ ↓ ↓ ↓ ↑ ↓ ↑ ↑ ↓ ↓ ↑ ↑ ↓

* * * * * *

1.6 2.4 1.5 1.5 1.6 −2.3 −8.1 −9.8 2.8 −2.2 2.4 5.3 −2.1 −3.7 1.8 3.7 −1.2

** **

* * *

**

*

**

*

**

*

a

17 metabolites were identified from the features that changed following BETX treatment, on the basis of mass accuracy and retention time. bThe up- (or down-) regulation of the arrow represents the relative increased (or decreased) concentration in the BETX-treated group as compared to the control group. *P-values less than 0.05. **P-values less than 0.01.

Table 2. MS/MS Transition and Parameters Used for the Detection of the Variations in the Contents of the Four Metabolites through Quantitative Analysis through Targeted Metabolomics name

formula

fragmentor (v)

collision energy (v)

Q1 mass (m/z)

Q3 mass (m/z)

dwell time (ms)

P-value

fold change

kynurenic acid kynurenine quinolinic acid 3-hydroxykynurenine

C10H7NO3 C10H12N2O3 C7H5NO4 C10H12N2O4

100 80 41 80

16 8 15 12

189.0 208.0 167.1 224.1

144 94 149.9 110

100 100 100 100

0.01 0.04 0.02 0.01

−2.1 1.8 3.0 2.1

of them were closely connected with each other. And the functions of this network involved in nervous system development and function, amino acid metabolism, and cellto-cell signaling and interaction, all of which were important for the toxicity of BETX. Moreover, the IPA software found an interesting signaling pathway, namely, the GABA receptor signaling pathway, as shown in Figure 4.

the difference between two groups, an OPLS model was constructed. The OPLS score plot demonstrated that control and BETX-treated groups were clearly distinguishable with the model parameters R2Y = 0.98, R2X = 0.56, and Q2 = 0.97 (Figure 3A). These parameters demonstrated that this model explained the data well. The S-plot showed the distribution of the potential biomarkers (Figure 3B). A higher distance of a triangle (a RT-m/z pair) from the origin represents a higher contribution on the classification of the groups. 200 significant variables (according to W*C and P(corr) in S-plot) contributing to the separation of two groups were selected. Then the 17 metabolites with fold changes larger than 1.5, Pvalues less than 0.05, and VIP values larger than 2.0 were identified as potential biomarkers (Table 1). Four metabolites, which may form part of the response of the nervous system, as determined through literature searches, were highlighted with a hatch pattern (Table 1).

BETX Inhibits IGABA in Rat Hippocampal Neurons

At a holding potential (VH) of −60 mV in the whole cell voltage clamping mode, an inward current was evoked in all of the tested neurons with the bath application of GABA (10 μM). The current was chloride-dependent and picrotoxin-sensitive, which indicated that it was the GABAAR-chloride channelsmediated current. As shown in Figure 5A, prior to the administration of BETX, GABA application leaded to a significant reduction of IGABA. The administration of 1, 3, and 10 μM BETX resulted in a percent inhibition of IGABA of 60.1 ± 6.7% (P < 0.001, n = 5) 36.2 ± 8.2% (P < 0.01, n = 6), and, 17.7 ± 3.4% (P < 0.05, n = 5), respectively (Figure 5B). Additionally, BETX effectively inhibited the IGABA induced by various GABA concentrations. The characteristics and interaction of the BETX-mediated inhibition of IGABA were shown in Figure S4 (Supporting Information).

Quantitative Analysis of Targeted Metabolomics

A quantitative analysis not only detects accurate delicate serum metabolic changes but also evaluates results of multivariate analysis.30 On the basis of the global profile, a total of four metabolites, namely, 3-hydroxykynurenine, quinolinic acid, kynurenic acid, and kynurenine, which were considered the most reliable and significant metabolites that were related to BETX-related cerebral lesions, were accurately quantified through a subsequent targeted metabolomics analysis. The accurate variations of these metabolites were shown in Table 2 (detailed in Table S1, Supporting Information). Bioinformatics analysis was performed to construct the biological association network of these four metabolites. The result showed that three

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DISCUSSION In general, naturally occurring polyacetylenes are widely distributed in food plants and herbs, such as carrots, celery, Bupleurum falcatum, Bupleurum spinosum, Bupleurum salicifolium, and Bupleurum acutifolium, and exhibit antibacterial, antiinflammatory, antifungal, and anti-platelet-aggregatory ef929

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compounds, BETX was isolated from B. longiradiatum. In agreement with the previous observation, the histopathological changes observed in this study demonstrate that BETX can induce internal brain injury in healthy mice. In addition, there were no obvious lesions in the heart, liver, and kidney, which suggested that the nervous tissue may be a specific target organ of BETX. To monitor the local metabolic changes in mice after BETX treatment, we used an LC−MS technique to profile the serum metabolites. The compendium of changes that we generated after the administration of BETX displayed many metabolic alterations. We identified 17 discriminating metabolites that exhibited perturbations in mice after BETX treatment. The quantitative analysis provided accurately subtle metabolic variations in serum that confirmed the findings from the metabolomic profiling. In the present study, targeted quantitative analysis was performed to accurately quantify four metabolites, namely, 3-hydroxykynurenine, quinolinic acid, kynurenic acid, and kynurenine. More details of networks affected by these four metabolites were analyzed with the IPA software. The primary biological function of the network was nervous system development and function, which showed consistency as expected. It was of great interest that the GABA receptor signaling pathway was found to associate with quinolinic acid, kynurenic acid, and kynurenine.34 Many types of neuronal cells exist in the nervous system, and they can be generally divided into two groups: inhibitory and excitatory neurons. The inhibitory neurons are mainly represented by GABAergic neurons.35 The interruption of the GABAergic processes exists in many neurological and psychiatric disorders, including emotional disorder, epilepsy, dyskinesia, such as sleep disorders, neuroplasticity, focal dystonia, stiff man syndrome, and drug and alcohol dependency.36 The functional defects of GABAergic signaling is a potentially conventional molecular mechanism for the epilepsy and comorbidity of autism.37 In the mammal brain, the GABA receptors are the main inhibitory neurotransmitter receptors. Those receptors exist in the postsynaptic membrane, which mediate short-term inhibition of neuronal signals, whereas those receptors in the extrasynaptic membrane are responsible for long-term inhibition.38 The imbalance of these neurons may also cause neurological disorders. Quinolinic acid, an endogenous metabolite, can lead to axonal lesions in the rat brain. It is also an N-methyl-D-aspartate (NMDA) receptor agonist biosynthesised from L-tryptophan and thus may cause NMDA neuronal injury and dysfunction. It may cause convulsions and excitotoxicity39,40 and can induce neuronal damage in the brain.41 The stimulation of GABA receptors potently inhibits quinolinic acid cytotoxicity on orexin neurons.42 In the substantia nigra, a major increase in GABA receptors was found after placement of a striatal quinolinic acid lesion.43 Kynurenic acid is a neuroprotective metabolite that exists in the brain at nanomolar neuroactive concentrations and is synthesized through the kynurenine pathway.41 This compound could protect the brain by acting as the antagonist of NMDA receptors.44,45 Kynurenic acid can control extracellular GABA levels in the rat striatum46 and decrease GABA release in a four-vessel occlusion rat model.47 Alteration of kynurenic acid was found in the amygdala together with a significant decrease of GABA.48 The kynurenine pathway, the key route of tryptophan metabolism, was found to be disturbed in brain function disorders, such as Alzheimer’s disease, acquired immunodeficiency syndrome (AIDS) demen-

Figure 4. Identification of the GABA receptor signaling pathway by IPA. The nodes represent the metabolites, and the lines between the nodes indicate the biological relationships between the two corresponding metabolites. Note that the colored symbols indicate the metabolites found in our study, whereas the transparent entries were molecules from the Ingenuity Knowledge Database. The red symbols represent the unregulated metabolites, and the green symbols represent the downregulated metabolites. The solid lines between the molecules indicate a direct physical relationship between the molecules, and the dotted lines indicate indirect functional relationships.

Figure 5. Inhibitory effect of BETX on IGABA in cultured hippocampal neurons. (A) 10 μM GABA-activated current (IGABA) in the absence and presence of 1, 3, and 10 μM BETX, respectively. (B) Concentration−response relationship for the BETX-mediated inhibition of IGABA. All of the currents were normalized to the peak amplitude activated by 10 μM GABA alone. Each point represents the mean ± SEM of the values obtained for 5−6 neurons. The continuous line shows the fit to the Hill equation.

fects.8,31,32 However, some polyacetylenes that are found at high concentrations in these plants are known to be neurotoxic.4 Previous studies have shown that the ethyl ether extract of B. longiradiatum exhibited high toxicity to mice, and its toxicity was correlated with the high content of polyacetylenes in this herb.33 To study the toxicity of these 930

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observed in the heart, liver, and kidney after BETX treatment. Figure S3. PCA model of QC samples showing the first two principal components: (blue ■) 6 QC samples, (black ■) BETX-treated group, (red ▲) control group. Figure S4. The characteristics and interaction of the BETX-mediated inhibition of IGABA. (A1−A4) Representative traces illustrating the effect of 3 μM BETX on IGABA activated by different concentrations of GABA. (B) Representative traces demonstrating the modulatory effect of BETX (3 μM) on IGABA using different drug application sequences (conditions b and c). Conditions a and d involve the induction of IGABA in the same neuron by GABA alone at the beginning and at the end of the experiment, respectively. (C) Means ± SEM of the percent inhibition of the peak amplitudes of IGABA obtained using different drug application protocols. The conditions were the same as those used in (B); n = 3. *P < 0.05 and **P < 0.001 compared with condition a. ##P < 0.01 compared as indicated. Table S1. Regression data and LLOQ of the analytes. This material is available free of charge via the Internet at http://pubs.acs.org.

tia complex, Huntington’s disease, malaria, cancer, depression, and schizophrenia.49 GABA was found to play a important role in the epilepsy triggered by kynurenines.50 The convulsant activity of kynurenine may be due to an interaction with GABA receptors.51 On the basis of the above discussions and the metabolite profiling results, we may draw the conclusion that GABA receptor signaling is one of the clues to study the toxicity of BETX. Furthermore, although it was not included in the network, 3-hydroxykynurenine also causes neuronal injury, gangrene, and apoptosis by generating reactive oxygen species, which can activate the kynurenine pathway by inducing the bioactivity of tryptophan-2,3-dioxygebase (TDO) and indoleamine-2,3-dioxygenase (IDO), which are the catalytic enzymes for the conversion from tryptophan to kynurenine.45 Thus, this metabolite is indirectly correlated with the GABA receptor. All together, the altered metabolites belonging to the GABA receptor signaling pathway may be a sign of BETX toxicity and reflect the abnormal GABA receptor signaling status in mice after BETX treatment. Our results could well supplement our understanding of BETX toxicity and provide insights for further explorations of the role of GABA receptor signaling in the toxicity mechanism of BETX. To improve the accuracy of our conclusion, we verified the results of the bioinformatics analysis using a cell line. Before the validation test, we found that various studies have focused on oenanthotoxin, which was an isomer of BETX. Oenanthotoxin has been found to change [K+]0 and [Ca2+]0 in oenanthotoxininduced epilepsy.52,53 Recent studies demonstrated that oenanthotoxin potently blocks GABAergic responses and that oenanthotoxin induces open-channel block and allosterically modulated GABAA receptors.54−56 Thus, we evaluated the influence of BETX on IGABA in cultured rat hippocampal neurons using a similar protocol. Our results show that BETX inhibits IGABA in a competitive manner, most likely by inhibiting GABAAR activation by interfering with the binding of GABA to its receptor. This finding was consistent with the bioinformatics analysis results and thus confirmed that toxic mechanisms can be effectively elucidated using metabolomics technology.

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Corresponding Authors

*Phone: (86)10 6406 7611. Fax: (86) 10 84032881. E-mail: [email protected]. *Phone: (86) 21 81871244. Fax: (86) 21 81871245. E-mail: [email protected]. Author Contributions #

Z. Zhang, C. Lu, and X. Liu contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work was supported by program NCET Foundation, NSFC(81230090), partially supported by Global Research Network for Medicinal Plants (GRNMP) and King Saud University, Shanghai Leading Academic Discipline Project (B906), Key laboratory of drug research for special environments, PLA, Shanghai Engineering Research Center for the Preparation of Bioactive Natural Products (10DZ2251300) and the Scientific Foundation of Shanghai China (12401900801, 09DZ1975700, 09DZ1971500, 10DZ1971700). National Major Project of China (2011ZX09307-002-03). National Key Technology R&D Program of China (2012BAI29B06).

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CONCLUSIONS This study demonstrates that four precise and quantitative metabolites, namely, 3-hydroxykynurenine, quinolinic acid, kynurenic acid, and kynurenine, induce fundamental changes in serum metabolite profile in response to BETX treatment, which strongly suggests that these four metabolites exert farreaching effects on the GABA signaling pathway, as determined through a bioinformatics analysis and validation test. These observations extend our understanding of the toxicity and bioactivity of polyacetylenes. Of further significance, this study will aid the hypothesis development and design of future therapeutic agents for the treatment of neurorelated disorders. In addition, our systematic study method can be used for the study on the toxicity mechanism of other toxic medical herbs.

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AUTHOR INFORMATION

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ABBREVIATIONS BETX, bupleurotoxin; LC−Q-TOF-MS, liquid chromatography quadruple time-of-flight mass spectrometry; IPA, Ingenuity Pathway Analysis; GABA, gamma-aminobutyric acid; HPLC− DAD−MS, high-performance liquid chromatography method coupled with diode array detector and mass spectrometry; ESI, electrospray ionization; PCA, principal components analysis; PLS-DA, partial-least-squares discriminate analysis; OPLS, orthogonal partial least-squares; EIC, extracted ion chromatograms; TIC, typical total ion current chromatograms

ASSOCIATED CONTENT

S Supporting Information *

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Figure S1. 13C NMR and 1H NMR spectrum (300 MHz, methanol-d4) of BETX. Figure S2. Histological evaluation of organs: (A) control heart; (B) BETX-treated heart; (C) control liver; (D) BETX-treated liver; (E) control kidney; and (F) BETX-treated kidney. The paraffin sections were stained with H&E. Scale = 100 μm. No histopathological abnormalities were

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