Integrated proteomics, biological functional assessments and

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Integrated proteomics, biological functional assessments and metabolomics reveal toosendanin induced hepatic energy metabolic disorders Xiaojing Yan, Yue Zhuo, Xiqing Bian, Jianmin Li, Yida Zhang, Lidong Ma, Guanghua Lu, Ming-Quan Guo, Jian-Lin Wu, and Na Li Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00350 • Publication Date (Web): 08 Mar 2019 Downloaded from http://pubs.acs.org on March 10, 2019

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Chemical Research in Toxicology

Integrated proteomics, biological functional assessments and metabolomics reveal toosendanin induced hepatic energy metabolic disorders

Xiaojing Yana,b, Yue Zhuoa, Xiqing Biana, Jianmin Lia, Yida Zhanga, Lidong Maa, Guanghua Luc, Ming-Quan Guod, Jian-Lin Wua,*, Na Lia,*

a

State Key Laboratory for Quality Research of Chinese Medicines, Macau University

of Science and Technology, Avenida Wai Long, Taipa, Macao b

Changzhou Affiliated Hospital of Nanjing University of Chinese Medicine, 25

Heping North Rd., Changzhou 213003, China c

School of Ethnic Medicine, Chengdu University of Traditional Medicine, Chengdu

611137, China d

Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture,

Wuhan Botanical Garden, Sino-Africa Joint Research Center, Chinese Academy of Sciences, Wuhan 430074, China

Corresponding authors. Email: [email protected]. Tel: +853 8897 2405. Fax: +853 2882 5886 (N Li); [email protected]. Tel: +853 8897 2406. Fax: +853 2882 5886 (JL Wu).

1 ACS Paragon Plus Environment

Chemical Research in Toxicology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

For TOC only


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Abstract Toosendanin (TSN), a compound from Melia toosendan, exerts severe hepatotoxicity, which restricts its clinical application. However, the mechanism is not clear. Our previous research found that covalent modification of TSN for proteins might be a possible reason using human liver microsome, and the glycolytic enzymes, triosephosphate isomerase 1 (TPIS) and α-enolase (ENOA), were responsible for the hepatotoxicity. In this study, we tried to prove them in cell and animal models by integration of proteomics, metabolomics and biological methods. Proteomics analysis in rats showed that TPIS and ENOA were covalently modified by TSN reactive metabolites (RMs). The biological functional assessments exhibited that the modifications inhibited the activity of TPIS and induced the activity of ENOA, respectively, in vitro and in vivo, followed by the increase of cellular methylglyoxal (MG), advanced glycation end products (AGEs) and reactive oxygen species (ROS)/superoxide, the induction of mitochondrial dysfunction, which further inhibited oxidative phosphorylation and stimulated glycolysis. Furthermore, metabolomics demonstrated the decrease of metabolites in tricarboxylic acid cycle, fatty acid β-oxidation and amino acid metabolism, i.e. TSN induced hepatocyte energy metabolism disorder. In conclusion, these data suggest novel mechanistic insights into TSN induced liver injury on the upstream level and provide valuable proteins and energy metabolic targets for diagnosis and therapy in clinic. 3 ACS Paragon Plus Environment


Keywords Toosendanin, hepatotoxicity, triosephosphate isomerase 1, -enolase, metabolic disorders


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1. Introduction

Toosendanin (TSN), a triterpenoid obtained from Melia toosendan Sieb et Zucc (Fig. 1A), is commonly used for the treatment of digestive tract parasitic diseases in 1, 2.

traditional Chinese medicine

Emerging evidence demonstrates that TSN has a

wide range of pharmacological activities including antibotulism effects inhibition

5-10.

However, TSN elicits highly hepatotoxic effect

11,

3, 4

and tumor

which seriously

restricts its clinical application. Previous studies uncovered that TSN induced hepatocellular mitochondrial dysfunction and caspase activation mainly through generation of reactive oxygen species (ROS) and mitogen-activated protein kinase (MAPK) activation in primary rat hepatocytes

11,

and the in vivo research showed

TSN-induced liver injury (TILI) may be caused by glutathione depletion, mitochondrial

dysfunction

and

microRNA-mRNA approach

12.

lipid

dysmetabolism

by

an

integrated

However, the upstream regulatory protein for

generation of ROS and MAPK and valuable protein targets for diagnosis and therapy in the clinic are still uncertain, which are urgently warranted to investigate. TSN contains a hepatotoxic furan ring and emerging evidence shows that furan-containing drug hepatotoxicity is related to the covalent binding of the reactive metabolites (RMs) to hepatic proteins

13-18.

In our previous investigation, 21 proteins

were found to be covalently modified by TSN RMs using human liver microsome and 5 ACS Paragon Plus Environment


a new online 2D-nano-LC-Q-TOF-MS method. Moreover, ingenuity pathway analysis (IPA) analysis indicated that the potential hepatotoxicity mainly focused on the glucose metabolism enzymes in glycolysis I, e.g. triosephosphate isomerase 1 (TPIS), -enolase (ENOA)

19.

Therefore, in this study, we attempted to determine whether

TSN RMs could modify the proteins in rat model and how to induce the hepatotoxicity by the modification. As a result, the covalent modification of TSN RMs for the energy metabolic enzymes, TPIS and ENOA, was one of possible reasons of hepatotoxicity. Furthermore, metabolomics were used to determine the hepatocellular consequence induced by TSN RMs modified proteins and provide the valuable targets for diagnosis and therapy in the clinic.

2. Materials and methods

2.1 Ethics statement

All animal experiments complied with the ARRIVE guidelines and were carried out in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments and under the license issued by the Macau SAR Government and approval of the “Institutional Animal Care and User Committee guidelines” of the Macau University of Science and Technology.

2.2 Chemicals 6 ACS Paragon Plus Environment

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Toosendanin (TSN) with a purity of over 98% was obtained from Chengdu MUST Bio-Technology

Co.,

Ltd.

Arachidonic

acid

(AA),

11(S)-,

12(S)-,

and

15(S)-hydroxyeicosatetraenoic acid (HETE), 12(S)-hydroxyeicosapentaenoic acid (HEPE), 15(S)-hydroxyeicosatrienoic acid (HETrE), 12(S)-hydroxyheptadecatrienoic acid (HHTrE) were purchased from Cayman Chemical (Ann Arbor, MI). Myristic acid, lauric acid, capric acid, octanoic acid, isooctanoic acid, caproic acid, isocaproic acid, malate, fumarate, α-ketoglutarate (α-KG), isocitrate, citrate and succinate, butyric acid, propionic acid, acetic acid, valeric acid and isovaleric acid, alanine, arginine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, tryptophan, glutamine,valine, citrulline, ornithine, and asparagine were purchased from Sigma-Aldrich Laboratories, Inc. (St. Louis, MO). 5-(Diisopropylamino)amylamine (HOBt),

(DIAAA),

1-hydroxybenzotriazole

hydrate

O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium

hexafluorophosphate (HATU), triethylamine (TEA) and DMSO were bought from Sigma-Aldrich Laboratories, Inc. LC-MS grade acetonitrile and HPLC grade methanol were obtained from Anaqua Chemicals Supply Inc., Ltd. (Houston, TA). Deionized water was prepared using a Millipore water purification system. DIAAA was purified by prepared HPLC before use. 7 ACS Paragon Plus Environment


2.3 Animal care and drug treatment

Sprague-Dawley rats (180-250 g) were purchased from the Chinese University of Hong Kong (Hong Kong) and shipped to Macau to conduct the experiment. The animals were housed in a 12 h light/dark cycles and temperature-controlled room and given ad libitum access to food and water. Rats were randomly divided into four experimental groups (n = 6). Rats in control group were treated with saline containing 1% dimethyl sulfoxide (DMSO) and 1% Tween 80 and the TSN groups were administered with the different doses of TSN (3.75, 7.5 and 15 mg/kg) through intraperitoneal injection once. After 24 h, the body weight was evaluated. The rats were anesthetized with nembutal sodium solution (50 mg/kg body weight), and the blood samples were collected from abdominal aorta. Then, the liver tissues were immediately perfused with 200 mL of ice-cold saline from abdominal aorta, weighted and then divided into three parts: the first part was fixed with 4% paraformaldehyde for staining with hematoxylin and eosin (HE), the second part was embedded in OCT for oil red staining, and the last part was frozen using liquid nitrogen and stored at -80 °C for proteomics and metabolomics analysis.

2.4 Assessment of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels


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The serum was obtained by centrifugation of rat blood at 1,500 rpm and 4 °C for 5 min. ALT and AST activities were tested using ALT and AST activity assay kits (Sigma, St Louis, MO). All procedures were performed according to the manufacturer’s instructions.

2.5 HE staining assay

Livers were fixed with 4% paraformaldehyde for at least 24 h and then embedded in paraffin, sectioned into 4 μm thick slices, stained with hematoxylin and eosin (HE), and finally evaluated by the pathologist in a blinded fashion with optical microscope.

2.6 Proteomics analysis by 2D SCX-nano-LC-Q-TOF-MS in rats

The adducted proteins were searched using our previous developed online 2D SCX-nano-LC-Q-TOF-MS approach 19. Rats were treated intraperitoneally with TSN at doses of 15 mg/kg. Liver were harvested at 1 h after the treatment. The liver tissues (0.1 g) were homogenized in 1.0 mL phosphate buffer (pH 7.4). After centrifugation at 9,000 g for 10 min, the supernatants (300 µL) were collected and mixed with 4 volumes of iced acetone. The samples were processed by centrifugation at 10,000 g for 5 min. The supernatants were collected and subjected to ultra-high performance liquid

chromatography-quadruple-time

of

flight

mass

spectrometry

(UHPLC-Q-TOF-MS) analysis in negative ion mode. At the same time, the 9 ACS Paragon Plus Environment


precipitates

were

collected

followed

by

digestion

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with

trypsin

and

2D

SCX-nano-LC-Q-TOF-MS analysis to search for the adducted proteins in Mascot. The samples without TSN administration were also prepared in parallel as control group.

2.7 Primary rat hepatocyte isolation and sandwich cell culture

Male SD rats (180-220 g) were anesthetized by intraperitoneal injection of nembutal sodium solution (50 mg/kg body weight) and the primary rat hepatocytes were isolated by using a two-step, type IV collagen perfusion as reported previously 20. The cells were plated on six-well dishes between two layers of soft-gel collagen I (sandwich) as described in 21, 22.

2.8 Cell viability analysis

Cell proliferation was assessed by MTT assay. Briefly, the primary hepatocyte cells were seeded in 96-well plates (BD Biosciences, North Ryde, Australia) at the density of 1 × 106 cells/mL. After 24-h incubation, the cells were treated with TSN at the concentration of 20, 40, 80, 160, 320 μM, respectively or medium for 24 h. Then, MTT (5 mg/mL) was added into the medium following incubation for 4 h at 37°C. The MTT medium was discarded and the cells were subsequently lysed using DMSO (150 μL/well). The optical density (OD) at 490 nm was measured using a 10 ACS Paragon Plus Environment

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Microplate Reader (SpectraMax, Molecular Device Co., Sunnyvale, CA, USA). All experiments were performed in triplicate and repeated at least three times. Data were calculated as the percentage of cell inhibition rate (%): [1-(Sample solution absorbance value/Control absorbance value)] × 100%. IC50 values were calculated using GraphPad Prism 5.0 software (GraphPad Software, Inc., San Diego, CA).

2.9 FLIPR assay for membrane potential

The primary hepatocyte cells were seeded in clear-bottom black 96-well plates (Corning, NY) at 1 × 105 cells/well. After grown to confluency, the medium was removed and replaced by 100 µL/well HBSS containing 20 mM HEPES to maintain the signal, and then 100 µL of blue-membrane potential dye was added into each well according to the manufacturer’s protocol. The membrane potential dye loaded cell plates were incubated for 30 min at 37 °C, and then the freshly prepared TSN in HBSS with 20 mM HEPES were added. The membrane potential was measured by Molecular Device's fluorometric imaging plate reader (FLIPR) as described. Relative changes in membrane potential fluorescence intensity (ΔF/F0) were calculated as the peak change in fluorescence amplitude (ΔF) divided by the initial intensity value (F0).

2.10 Enzymatic activities of TPIS and ENOA

The enzymatic activities of TPIS and ENOA protein were determined using TPIS 11 ACS Paragon Plus Environment


(Biovision) and ENOA (Sigma) activity assay kits, respectively. Cells were seeded in 6-well plates at a density of 1×106 cells/well for 24 h and then incubated with TSN at the concentration of 20, 40 and 80 μM for another 24 h. Treated cells were washed with cold PBS and lysed using 100 μL TPIS and ENOA assay buffer, respectively, and then the supernatants were collected and the activities of TPIS and ENOA were detected by corresponding kits according to the instructions.

2.11 Western blot assay

Cell or tissue extracts were prepared in RIPA buffer (CST, Boston, MA) containing complete protease inhibitor and phosphatase inhibitor (Roche, CA). Protein concentrations were quantified using BCA protein assay kit (Bio-Rad, CA) following the manufacturer's instructions. Then proteins were resolved on SDS-PAGE gels and electrophoretically transferred to a PVDF membrane (Millipore, Burlington, MA). The membranes were incubated with primary antibodies overnight at 4 °C, followed by incubation with appropriate secondary antibodies at 1:10,000 dilution (CST, Boston, MA) for 1 h. Signals were developed using ECL chemiluminescence detection reagents (Santa Cruz, CA) and visualized on Amersham Imager 600 system (GE).

2.12 Lactate and pyruvate production assay


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Lactate and pyruvate production in primary hepatocyte cells was measured with the Lactate and Pyruvate Assay Kit (Biovision, San Francisco, CA) using an enzymatic reaction according to the manufacturer’s instructions.

2.13 Oxygen consumption and Glycolysis assay

Cellular oxygen respiration and glycolysis in primary hepatocyte cells were measured with Seahorse XFp Extracellular Flux Analyzer (Seahorse Biosciences, North Billerica, MA) as described previously

23.

Cells were seeded in Seahorse XFp cell

culture miniplate at 1 × 105 cells/well in medium and then incubated for 24 h with TSN at the concentration of 40 μM. Before assessing the cellular functions, the medium was replaced with 750 μL Seahorse medium and equilibrated at 37 °C in a CO2-free incubator for 30 min. Plates were transferred to the XFp analyzer. The basal oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were determined using Seahorse XFp Cell Mito Stress and Glycolysis Stress Test kit.

2.14 Methylglyoxal (MG) measurement

The cellular MG in hepatocyte tissues was quantified by UHPLC-Q-TOF-MS 24, 25. In brief, liver tissues were homogenized in cold PBS by sonication (30 s, three times) and incubated with 0.45 N perchloric acid at room temperature to remove the proteins. Then, the supernatant reacted with 10 mM o-phenylenediamine (o-PD) (Sigma) at 13 ACS Paragon Plus Environment


room temperature in the dark for 3 h. The produced quinoxaline derivative, 2-methylenediamine (2-MG), was quantified by UHPLC-Q-TOF-MS system equipped with a BEH C18 column (2.1 × 100 mm, 1.7 µm) (Waters, Milford, MA). The mobile phase was composed of A (0.1% formic acid-containing water) and B (0.1% formic acid-containing acetonitrile) with a gradient elution: 5%-50% B for 0-4.5 min, 50%-95% B for 4.5-6.5 min. The flow rate was 0.35 mL/min, and the column temperature was maintained at 40 °C.

2.15 Elisa assay for rat Advanced Glycation End Products (AGEs)

The rat serum and primary hepatocyte cell supernatant were collected and detected by Rat AGEs Elisa kit (CUSABIO) according to the manufacturer’s instructions. The optical density of each well was determined using a microplate reader at 450 nm.

2.16 Reactive oxygen species and superoxide assay

Primary hepatocyte cells seeded in black clear-bottom 96-well imaging plates and incubated with TSN at the concentration of 20, 40 and 80 μM for 24 h. Treated cells were washed with PBS twice and stained with ROS/superoxide kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions. Images were captured by an In Cell Analyzer 6000 system (GE Healthcare). Staining intensity was detected by Microplate Reader (SpectraMax, Molecular Device Co., Sunnyvale, CA). 14 ACS Paragon Plus Environment

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2.17 HCS analysis for mitochondrial membrane potential (ΔΨm)

Primary hepatocyte cells seeded in black clear-bottom 96-well imaging plates and incubated in TSN at the concentration of 20, 40 and 80 μM for 24 h. Treated cells were washed with PBS twice and stained with Rhodanmin 123 (Rh 123) (Thermo Fisher Scientific Inc., MA) for 30 min, respectively. After washing with PBS, cells were fixed with 4% paraformaldehyde for 15 min and washed twice with PBS, labeled with DAPI for 20 min, followed by washing with PBS. Then the plates were sealed before adding PBS (100 µl/well). Images were captured by an In Cell Analyzer 6000 system (GE Healthcare). Staining intensity was calculated from captured images using the In Cell Analyzer 1000 workstation software (GE Healthcare).

2.18 Metabolomic analysis

The metabolites were analyzed using our previous developed UHPLC-Q-TOF-MS approach 26. The rat serum samples were mixed with 4 volumes of cold methanol to remove the proteins by centrifugation at 13,000 rpm for 5 min at 4 °C. The extractions were repeated 3 times and the combined supernatants were dried under a nitrogen stream. Next, the residue was derivatized as follows

26.

Samples and standards were

sequentially mixed with 5 μL of 20 mM HOBt in DMSO, 5 µL of 100 mM DIAAA in DMSO containing 200 mM TEA, and 5 µL of 200 mM HATU in DMSO, followed by



1 min incubation at room temperature. Finally, 35 µL acetonitrile was added to make up to the final volume of 50 µL, and 1 µL was directly injected into UHPLC-Q-TOF-MS and analyzed in positive ion mode. The metabolites were identified by the comparison with the corresponding standards.

2.19 Statistical analysis

Results were expressed as mean ± SD. Data were analyzed using SPSS 17.0 software (SPSS Inc., Chicago, IL). The significance of difference was determined by one-way ANOVA with LSD tests, and Student’s t-test was used for two groups’ comparison. Values of P < 0.05 were considered to be statistically significant. Western images were quantified by densitometry using Image J software (National Institutes of Health).

3. Results

3.1 TSN induces hepatotoxicity in vivo

The hepatotoxicity of TSN was first assessed using SD rats. TSN obviously decreased the body weight and slightly increased ALT and AST levels in the serum in a concentration-dependent manner compared with control group (P < 0.05) (Fig. S1A-B). Besides, histopathology of liver tissues also revealed cell swelling, cytoplasmatic vacuoles, inflammatory foci and hydropic degeneration, suggesting that 16 ACS Paragon Plus Environment

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TSN treatment induced serious liver injury (Fig. 1B). Notably, the hallmark of this injury, cytoplasmatic vacuoles, is an accumulation of microvesicular fat in hepatocytes and even can evolve into macrovesicular fatty liver with focal necrosis (Fig. 1C).

3.2 TSN induces hepatotoxicity in vitro

TSN also exhibited evident cytotoxicity on primary hepatocyte cells. As shown in Fig. 1D, TSN significantly inhibited rat primary hepatocyte cell viability in a concentration-dependent manner compared with control group (P < 0.05) and the IC50 value was 30.65 μM. Importantly, the results also displayed the significant increase of cell membrane potential at 60 s and decrease of cell membrane potential at 3000 s compared with control group (P < 0.001) at the concentration of 80-360 μM (Fig. S2A-C), which indicated that TSN could promote the Na+ influx and K+ efflux, shrink cell membrane and increase cell membrane permeability.

3.3 TSN RMs covalently modify TPIS and ENOA in rats

After administration, TSN was detected from the rat liver at 7.463 and 7.612 min using UHPLC-Q-TOF-MS by comparison with authentic standard, while no corresponding peaks were observed in control group (Fig. S3A). At the same time, a metabolite of TSN was also detected at 7.130 and 7.263 min (Fig. S3B) and identified 17 ACS Paragon Plus Environment


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as the diesterolysis product of TSN from the pseudo-molecular ion at m/z of 489.2104 (C26H34O9) and MS/MS characterization compared to TSN (Fig. S3C-D). It has been determined from TSN treated rats using MS and NMR27. Due to the presence of a hemiacetal group, TSN and its diesterolysis product showed two peaks, respectively, in current LC-MS condition. Next, the modification of TSN RMs for liver proteins of rats was determined using 2D SCX-nano-LC-Q-TOF-MS analysis

19.

Considering the diesterolysis product of

TSN might also modify lysine residues in cellular proteins, the molecular compositions of C26H32O9 were added to the lysine residues in Mascot as variable modifications. At least 2 proteins were found to be covalently modified by RMs of diesterolysis product of TSN. The unique peptide of

20KCLGELICTLNAAK33

for

protein TPIS showed a molecular ion [M+2H]2+ at m/z 982.9973, which was 488 Da more than that of the corresponding non-modified peptide; therefore, its lysine residue should be modified by RM of diesterolysis product of TSN (Fig. S4A). The molecular formula of TSN-RM adduct is 2H less than the sum of diesterolysis product and peptide. Moreover, it has been reported that the TSN forms covalent bond with lysine by the addition of an amino group to furan residue in TSN by NMR27, and the similar combination mode also has been proved by the fragmentation ions in our previous paper19. In addition, the molecular formula of the TSN adducts mentioned above were 18 ACS Paragon Plus Environment

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also 2H less than the sum of TSN and lysine. Thus, the diesterolysis product of TSN could modify lysine residues in proteins in the same way. The binding site was deduced to be K20 from the same mass increase of 488 Da in the fragmentation ions of b3-b10 and b13 of the aged peptide (Fig. S4B). Similarly, RM of diesterolysis product of TSN was found to covalently connect with K126 of ENOA (Fig. S4C).

3.4 TSN inhibits TPIS enzyme activity but does not regulate protein level in vivo and in vitro

TPIS plays an important role in glycolysis and the deficiency is associated with the accumulation of MG and AGEs

28.

The accumulation of glycation adducts results in

oxidative stress, DNA damage and apoptosis

29.

As shown in Fig. 2A-B, TSN

exposure significantly suppressed TPIS activity in vivo and in vitro compared with control group (P < 0.05). However, TSN did not affect the protein level of TPIS (P > 0.05) in vivo and in vitro (Fig. 2C-D), suggesting that TSN affected TPIS functions mainly through covalent modification for TPIS protein and further inhibiting its activity.

3.5 TSN increases cellular MG, AGEs and ROS/superoxide levels

Subsequently, the cellular MG was found to be markedly elevated in TSN group analyzed by LC-MS method (P < 0.05) (Fig. 3A). Further, AGEs and 19 ACS Paragon Plus Environment


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ROS/superoxide levels were detected by rat AGEs Elisa and ROS/superoxide kits, and the AGEs levels were obviously increased in rat serum (Fig. 3B) and primary hepatocyte cell supernatant (Fig. 3C) (P < 0.01), and the ROS/superoxide levels were promoted in primary rat hepatocytes compared with control group (P < 0.05) (Fig. 3D-E).

3.6 TSN inhibits mitochondrial membrane potential (ΔΨm) and induces cell apoptosis

Accumulated ROS could induce apoptosis and are normally accompanied by a decreased mitochondrial membrane potential (ΔΨm), which is essential for ATP production, further contributing to the decreased energy production

30, 31.

To

determine the effects of TSN on mitochondrial function, the cell ΔΨm was firstly detected. Compared with control group, the cell nuclear area and ΔΨm fluorescent intensity were significantly decreased (Fig. 4A-C) when cells were exposed to TSN for 24 h in a concentration-dependent manner (P < 0.01). The results suggested that TSN could promote primary rat hepatocyte cell mitochondrial dysfunction and further induce mitochondrial-mediated cell apoptosis.

3.7 TSN promotes ENOA enzyme activity but does not modulate protein level in vivo and in vitro

ENOA, an essential glycolytic enzyme, can catalyze 2-phosphoglycerate to 20 ACS Paragon Plus Environment

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phosphoenolpyruvate, stimulate glycolysis, and promote the formation of lactate and pyruvate

32.

To evaluate the effects of TSN on ENOA protein, the activity kit and

western blot were used to detect the ENOA activity and protein level, respectively. The significant increase of ENOA activity was induced by TSN in vivo and in vitro (Fig. 5A-B) (P < 0.05). Strikingly, TSN exhibited no obvious influence on ENOA protein level compared with control group (P > 0.05) (Fig. 5C-D), which indicated that TSN affected ENOA functions mainly through covalent modification for ENOA protein and further increasing its activity.

3.8 TSN stimulates glycolysis and inhibits mitochondrial oxygen consumption

The lactate and pyruvate concentration in primary rat hepatocytes were obviously increased in TSN treated group compared with control group (P < 0.001) (Fig. 6A-B). Further, to verify the effects of TSN on glycolysis, the Seahorse XFp Extracellular Flux Analyzer was used to determine the glycolysis and cellular oxygen respiration in primary rat hepatocytes. As shown in Fig. 6C, TSN significantly stimulated glycolysis compared with control group, which should be through binding ENOA protein and increasing ENOA activity. Besides, OCR results showed that TSN markedly suppressed cellular oxygen respiration and ATP production, which indicated that TSN could inhibit mitochondrial oxidative phosphorylation (Fig. 6D).



3.9 TSN induces hepatocytes energy metabolism disorder

To further determine if alterations of TSN RMs-adducted proteins (TPIS and ENOA) are accompanied by changes in respective metabolic pathways, the metabolites in rat serum were analyzed by UHPLC-Q-TOF-MS. Glycolysis is the cytosolic pathway that converts glucose to pyruvate. Under aerobic conditions, pyruvate enters the mitochondria to be oxidized by pyruvate dehydrogenase and enzymes of the TCA cycle

33.

Thus, TCA cycle plays an important role in mitochondrial metabolism. As

shown in Fig. 7A, there was a significant decrease in TCA cycle related metabolites, α-KG and fumarate, in TSN treated group compared with control group (P < 0.05). Moreover, we also found that TSN markedly inhibited the amounts of long-chain unsaturated free fatty acid, arachidonic acid (P < 0.01), and its oxidation products (11(S)-, 15(S)-HETEs) and metabolites (12(S)-HHTrE) (Fig. 7B). Besides, TSN obviously increased the long-chain saturated fatty acid, myristic acid, while decreased 9 saturated free fatty acids including the degradation products of myristic acid β-oxidation, lauric acid, capric acid, octanoic acid, caproic acid, butyric acid and acetic acid, compared with the control group (P < 0.05) (Fig. 7C), suggesting that TSN could inhibit long-chain unsaturated and saturated free fatty acid β-oxidation (FAO). Reducing lipid oxidation is highly correlated with hepatic steatosis 34, which indicated that TSN may further induce hepatic steatosis. Besides, amino acids 22 ACS Paragon Plus Environment

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metabolism requires mitochondria participation, and the significant decrease of asparagine, lysine, threonine and methionine (Fig. 7D) suggested hepatic mitochondrial dysfunction as a critical reason in TSN induced hepatotoxicity. In conclusion, TSN induced hepatocytes energy metabolomics disorder (Fig. 8), which indicated that TSN could induce mitochondrial dysfunction and may cause hepatic steatosis.

4. Discussion

Drug induced liver injury (DILI) has emerged as a significant clinical health problem with economic impact on the post-marketing withdrawal of new medications, which urgently requires expansion of basic research into the mechanism of DILI and preclinical prediction for hepatotoxicity

35, 36.

The mechanisms of DILI usually

involves the participation of a toxic drug or drug RMs that promotes a variety of chemical reactions, such as the depletion of reduced glutathione; covalently binding to proteins, lipids, or nucleic acids; or inducing lipid peroxidation

14, 17.

However, the

mechanisms involved in the protein adduct-induced hepatotoxicity are largely uncertain and the clinical consequences of drug bioactivation and covalent modification for proteins are unpredictable. Excitedly, our previous study provided a valuable method to predict the potential mechanisms for DILI on the upstream level in human liver microsome 19. Therefore, the aim of the current study was to verify the 23 ACS Paragon Plus Environment


Page 24 of 49

adducted proteins by TSN RMs in rats and better understand the functional and cellular consequences of protein adduct formation by TSN RMs in vitro and in vivo. Besides, we will provide valuable metabolic targets for diagnosis and therapy in clinic by using metabolomics. Firstly, the hepatotoxicity was investigated using in vitro and in vivo systems. Strikingly, accumulation of microvesicular fat in hepatocytes described that TSN may induce liver steatosis. Our previous results from human liver microsome indicated that the top pathways containing two key metabolic enzymes (TPIS and ENOA) might be responsible for TILI. Thus, subsequently, proteomics analysis was conducted to verify whether the metabolic enzymes were modified by TSN RMs in vivo. The results demonstrated that the glycolytic enzymes of TPIS and ENOA were also covalently adducted by TSN RMs in rats. Therefore, we designed to determine the impacts of TSN RMs modification on protein activity, level and functions in vitro and in vivo. TPIS functions at a metabolic cross-road ensuring the rapid equilibration of the triosephosphates produced by aldolase in glycolysis. The impairment of TPIS activity results in accumulation of dihydroxyacetone phosphate followed by its chemical conversion into the toxic MG, leading to the formation of AGEs and increase of ROS 37.

The accumulation of ROS could further induce mitochondrial dysfunction

inhibit TCA cycle and stimulate glycolysis

39-42.

38,

According to the results, TSN


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obviously inhibited TPIS activity and did not regulate TPIS protein level, suggesting that TSN was the TPIS enzyme inhibitor and affected TPIS functions mainly through covalently targeting TPIS protein and further inhibiting its activity. Subsequently, markedly increase of MG, AGEs and ROS/superoxide were found by TSN treatment. Furthermore, our data also suggested that TSN promoted hepatocyte cell mitochondrial dysfunction and further induced mitochondrial-mediated cell apoptosis. Taken together, these data confirm that deficiency of hepatic TPIS activity induced by TSN lead to the overproduction of toxic MG, AGEs and ROS, and not only further promote mitochondrial dysfunction and induce mitochondrial-mediated cell apoptosis, but also may inhibit TCA cycle and stimulate glycolysis. Besides, mitochondrial dysfunction due to long-chain Acyl-CoA dehydrogenase deficiency causes hepatic steatosis

43,

which indicated that TSN may promote hepatic steatosis through

inhibiting TPIS activity and further inducing mitochondrial dysfunction. ENOA, a key glycolytic enzyme, is critical for cellular energy metabolism, which generates ATP during glycolysis

44, 45.

TSN, as an ENOA enzyme accelerator,

significantly increased ENOA activity while its protein level was not altered, indicating that TSN affected ENOA functions mainly through covalently adducting ENOA protein, thereby further increasing ENOA activity. Lactate and pyruvate are the end products of glycolysis

46.

Supportive evidence for a significant increase in 25

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Page 26 of 49

lactate and pyruvate concentration in TSN group was detected, and Seahorse XFp Extracellular Flux Analyzer showed that TSN significantly stimulated glycolysis and markedly suppressed cellular oxygen respiration and ATP production, suggesting that TSN may stimulate glycolysis and inhibit mitochondrial function thereby reducing TCA cycle and oxidative phosphorylation. This may ultimately lead to hepatic failure with energy metabolism disorder and lactic acidosis. In order to further confirm the effects of the key adducted proteins (TPIS and ENOA) by TSN RMs on energy metabolic pathway and provide valuable metabolic targets for diagnosis and therapy in clinic, metabolomics was conducted. Mitochondrial TCA cycle is a core pathway for the metabolism of sugars, lipids and amino acids

47.

It was found that TSN caused a strong decrease of TCA cycle

intermediates such as α-KG and fumarate in rat serum, indicating that TSN promoted the reduction of mitochondrial energy metabolism. The lipid content of hepatocytes is regulated by the integrated activities of cellular enzymes that catalyze lipid uptake, synthesis, oxidation, and export. When “input” of fats exceeds the capacity for fatty acid oxidation or export, then hepatic steatosis occurs

48.

Therefore, toxins impaired

hepatic mitochondrial fatty acid oxidation will inhibit the elimination of fat from the liver and is strongly associated with hepatic steatosis. Arachidonic acid (AA), as a long-chain unsaturated fatty acid, frequently decreased in non-alcoholic fatty liver 26 ACS Paragon Plus Environment

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disease (NAFLD) in patients

49, 50.

Our data revealed the significant decrease of AA

and its oxidation products and metabolites in TSN RMs treated group. Moreover, TSN treatment resulted in a markedly decrease in the long-chain saturated free fatty acids β-oxidation. Besides, we also observed an obvious reduction in various amino acids induced by TSN, like asparagine, lysine, threonine and methionine in rat serum, because TCA cycle is a major pathway for amino acids metabolism. Collectively, data indicated that TSN inhibition on metabolites in TCA cycle, FAO and amino acid metabolism can further promote hepatic mitochondrial energy metabolism disorder and hepatic steatosis, supporting the prediction for TSN induced potential DILI from proteomics and proving that mitochondrial energy metabolism disorder and hepatic steatosis are critical event in TSN hepatotoxicity. Importantly, the decrease of TCA cycle, FAO and amino acid metabolism demonstrated by metabolomics suggested valuable metabolic targets for diagnosis and therapy of drug-induced mitochondrial injury in clinic. 5. Conclusion In summary, proteomics in rats indicated that the glycolytic enzymes of TPIS and ENOA were covalently targeted by TSN RMs. Moreover, TSN RMs was verified as TPIS enzyme inhibitor and ENOA enzyme accelerator and affected their activities and functions in vitro and in vivo. Further, metabolomics data suggested that energy 27 ACS Paragon Plus Environment


metabolism disorder and hepatic steatosis are the critical event in TSN induced hepatotoxicity and the key modified energy enzymes (TPIS and ENOA) are mainly responsible for TILI (Fig. 9). Our results provided mechanistic insights into TILI. More importantly, the integrated methods of proteomics and metabolomics are established to predict and verify the potential mechanisms for TILI on the upstream level and provide valuable protein and energy metabolic targets for diagnosis and therapy of toxic complex drug induced mitochondrial injury in clinic.

Conflict of interest

The authors declared that they have no conflict of interest.

Funding

This work is supported by Macao Science and Technology Development Fund (003/2016/A1) and National Natural Sciences Foundation of China (81603296). Supporting Information Supplemental Table 1: Identified metabolites from sera of rats dosed with toosendanin Supplemental Figure 1: Effects of TSN on the rate of body weight change and ALT and AST levels Supplemental Figure 2: Effects of TSN on the primary hepatocyte cell membrane potential 28 ACS Paragon Plus Environment

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Supplemental Figure 3: Extracted ion chromatograms (EICs) of TSN (A) and its diesterolysis product (B) in rat livers, their MS/MS spectra (C and D), and the structures of diesterolysis product of TSN (E) Supplemental Figure 4: Covalent modification on K20 of TPIS and K126 of ENOA

Acknowledgements

We thank Leong Soi leng and Charles Ho for HE staining and frozen slicing in this study (Department of Pathology, University Hospital, Taipa, Macau). Abbreviations AA: Arachidonic acid; AGEs: Advanced glycation end products; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; DILI: Drug-induced liver injury; ECAR: Extracellular acidification rate; FAO: Fatty acid β-oxidation; FLIPR: Fluorometric

imaging

plate

reader;

HATU:

O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate; HCS :

High

content

screening;

HE:

Hematoxylin

and

eosin;

HEPE:

Hydroxyeicosapentaenoic acid; HETE: Hydroxyeicosatetraenoic acid; HETrE: Hydroxyeicosatrienoic 1-Hydroxybenzotriazole

acid;

HHTrE:

hydrate;

Hydroxyheptadecatrienoic

IPA:

Ingenuity

pathway

acid;

HOBt:

analysis;

α-KG:

α-Ketoglutarate; MAPK: Mitogen-activated protein kinase; MG: Methylglyoxal; NAFLD: Non-alcoholic fatty liver disease; OCR: Oxygen consumption rate; RMs: 29 ACS Paragon Plus Environment


Reactive metabolites; ROS: Reactive oxygen species; TCA: Tricarboxylic acid; TEA: Triethylamine; TILI: TSN-induced liver injury; UHPLC-Q-TOF-MS: Ultra-high performance liquid chromatography-quadrupole-time of flight mass spectrometry; ΔΨm: Mitochondrial membrane potential

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Figure Legends

Fig. 1 TSN induced hepatotoxicity in vivo and in vitro. (A) Structure of toosendanin (TSN). (B) Photomicrographs of rat liver tissues were stained with hematoxylin and eosin (HE) (200×). (C) TSN induced hepatocytes cytoplasmatic vacuoles (400×). (D) TSN inhibited cell proliferation on rat primary hepatocyte cells. Values expressed as mean ± SD from three independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001 vs. control group.



Page 42 of 49

Fig. 2 Effects of TSN on enzyme activity and protein expression. TSN inhibited TPIS enzyme activity in vivo (A) and in vitro (B). The protein levels of TPIS in rats (C) and primary hepatocyte cells (D) were assayed by western blot. Values expressed as mean ± SD from three independent experiments, *P < 0.05, control group.


**P

< 0.01,

***P

< 0.001 vs.

Page 43 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig. 3 Effects of TSN on cellular MG, AGEs and ROS/superoxide. (A) The cellular MG level in rat hepatocyte tissues was detected by LC-MS analysis. The AGEs level in rat serum (B) and primary hepatocyte cell supernatant (C) were detected by Rat AGEs Elisa kit. The ROS/superoxide kit was used to assess the cellular ROS (D) and superoxide (E) level in primary hepatocyte cells. Values expressed as mean ± SD from three independent experiments, *P < 0.05,

**P

< 0.01,

group.


***P

< 0.001 vs. control


Fig. 4 Effects of TSN on mitochondrial membrane potential (ΔΨm). (A) The mitochondrial membrane potential (ΔΨm) was detected by In Cell analyzer 1000 system in primary hepatocyte cells. (B) The nucleus area and (C) ΔΨm fluorescent intensity was calculated by In Cell analyzer 1000 workstation software. Values expressed as mean ± SD from three independent experiments, *P < 0.05, **P < 0.01, ***P

< 0.001 vs. control group.


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Fig. 5 Effects of TSN on ENOA enzyme activity and protein level. TSN promoted ENOA enzyme activity in vivo (A) and in vitro (B). The protein levels of ENOA in rats (C) and primary hepatocyte cells (D) were assayed by western blot. Values expressed as mean ± SD from three independent experiments, *P < 0.05, **P < 0.01, ***P

< 0.001 vs. control group.



Fig. 6 Effects of TSN on lactate, pyruvate, glycolytic rate (ECAR) and oxygen consumption rate (OCR). The lactate (A) and pyruvate (B) production levels in primary hepatocyte cell medium. The ECAR (C) and OCR (D) in primary hepatocyte cells determined by using a Seahorse XFp analyzer after exposure to TSN for 2 h. Values expressed as mean ± SD from three independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001 vs. control group.


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Fig. 7 Effects of TSN on energy metabolism. (A) The tricarboxylic acid (TCA) cycle, (B) long-chain unsaturated free fatty acids (LCUFFAs), (C) saturated free fatty acids (SFFAs) and (D) amino acids in rat serum were analyzed by UHPLC-QTOF/MS. Values expressed as mean ± SD from three independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001 vs. control group.



Fig. 8 Changes of metabolites in different metabolic pathways induced by TSN. TSN inhibited mitochondrial TCA cycle related metabolites, long-chain unsaturated and saturated free fatty acids β-oxidation (FAO) and amino acids metabolism that required mitochondria participation, which finally induced hepatocytes mitochondrial energy metabolomics disorder.


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Fig. 9 The proposed mechanism for TILI through modification of the metabolic enzymes (TPIS and ENOA). TSN covalently targeted ENOA and accelerated its activity, further stimulated glycolysis and promoted pyruvate and lactate levels, and finally induced hepatic lactic acidosis. Moreover, TSN covalently binding to TPIS and inhibited its activity, further accumulated cellular MG, AGEs and ROS, which stimulated glycolysis, caused mitochondrial dysfunction and suppressed TCA cycle, oxidative phosphorylation, FAO and amino acid metabolism, finally induced mitochondrial energy metabolism disorder.


Integrated proteomics, biological functional assessments and

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