astin b

8/10/2019 Astin B

1/9

Astin B, a cyclic pentapeptide from Aster tataricus, induces apoptosis

and autophagy in human hepatic L-02 cells

Li Wang, Ming-Dan Li, Pei-Pei Cao, Chao-Feng Zhang , Fang Huang, Xiang-Hong Xu,Bao-Lin Liu, Mian Zhang

Research Department of Pharmacognosy, China Pharmaceutical University, Longmian Road 639, Nanjing 211198, PR China

a r t i c l e i n f o

Article history:

Received 15 February 2014

Received in revised form 21 August 2014

Accepted 4 September 2014

Available online 16 September 2014

Keywords:

Astin B

Liver injury

Apoptosis

Autophagy

Aster tataricus

a b s t r a c t

Astins (including astin B) are a class of halogenated cyclic pentapeptides isolated from the medicinal herb

ofAster tataricus. However, our previous works showed that the herbal medicine was hepatotoxic in vivo,

and a toxicity-guided isolation method led to the identification of a cyclopeptide astin B. Astin B is struc-

turally similar to cyclochlorotine, a well-known hepatotoxic mycotoxin. Thus, the aim of this study was

to determine the potential cytotoxic effects and the underlying mechanism of astin B on human normal

liver L-02 cells. We found that astin B has hepatotoxic effects in vitro and in vivoand that hepatic injury

was primarily mediated by apoptosis in a mitochondria/caspase-dependent manner. Astin B provoked

oxidative stress-associated inflammation in hepatocytes as evidenced by increased levels of reactive oxy-

gen species (ROS), reduced contents of intracellular glutathione (GSH), and enhanced phosphorylation of

c-Jun N-terminal kinase (JNK). Furthermore, the mitochondria-dependent apoptosis was evidenced by

the depolarization of the mitochondrial membrane potential, the release of cytochrome c into cytosol,

the increased ratio of Bax/Bcl-2, and the increased activities of caspases-9 and -3. Interestingly, astin B

treatment also induces autophagy in L-02 cells, characterized by acidic-vesicle fluorescence, increased

LC3-II and decreased p62 expression. Autophagy is a protective mechanism that is used to protect cells

from apoptosis. The presence of autophagy is further supported by the increased cytotoxicity and the

enhanced cleaved caspase-3 after co-treatment of cells with an autophagy inhibitor, also by increasedLC3-II and decreased p62 after co-treatment with a caspase inhibitor. Taken together, astin B, most likely

together with other members of astins, is the substance that is primarily responsible for the hepatotox-

icity ofA. tataricus.

2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

The hepatocyte is especially vulnerable to injury due to its cen-

tral role in xenobiotic metabolism including the metabolism of

drugs. Therefore, drug-induced liver injury is the most frequent

reason for post-marketing warnings and withdrawal[1]. The death

of hepatocytes and other types of hepatic cells is a characteristic

feature of drug-induced injury[2]. Based on morphological appear-

ance, cell death has been classified into several modes such as

apoptosis, necrosis, necroptosis, autophagy, and cornification [3].

Of these, apoptosis is considered to be a common pathway for

execution of hepatocytes upon liver injury. In response to xenobi-

otic metabolism, hepatocytes often generate excess reactive

oxygen species (ROS), which evoke oxidative stress-associated

inflammation, leading to mitochondrial dysfunction [4,5].

Mitochondria play a key role in the regulation of redox homeosta-

sis and apoptosis in cells [6]. A loss of mitochondrial function

allows the release of a number of proapoptotic factors, such as

cytochrome c, which induces caspase activation to trigger

mitochondria-dependent apoptotic cell death [7]. Accumulating

evidence demonstrates that hepatocyte apoptosis is tightly associ-

ated with drug-induced liver injury [6,8]. However, autophagy is

understood to be a mechanism of protection against various forms

of human diseases, including drug-induced liver injury, with an

extremely complex interplay[9,10].

Astins, mainly including astins AI, are a class of natural haloge-

nated cyclic pentapeptides isolated from the root ofAster tataricus

L. f. (RAT, Compositae) that has been used for over 2000 years in

traditional Chinese medicine for the relief of coughs and the

removal of phlegm. This class of compounds exhibits antitumor

and immunosuppressive activities[1113]. However, our previous

studies discovered the potent hepatotoxicity of RAT in mice, and a

http://dx.doi.org/10.1016/j.cbi.2014.09.003

0009-2797/ 2014 Elsevier Ireland Ltd. All rights reserved.

Corresponding authors. Tel./fax: +86 25 86185137.

E-mail addresses: [email protected] (C.-F. Zhang), [email protected]

(M. Zhang).

Chemico-Biological Interactions 223 (2014) 19

Contents lists available at ScienceDirect

Chemico-Biological Interactions

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / c h e m b i o i n t
http://dx.doi.org/10.1016/j.cbi.2014.09.003mailto:[email protected]:[email protected]://dx.doi.org/10.1016/j.cbi.2014.09.003http://www.sciencedirect.com/science/journal/00092797http://www.elsevier.com/locate/chembiointhttp://www.elsevier.com/locate/chembiointhttp://www.sciencedirect.com/science/journal/00092797http://dx.doi.org/10.1016/j.cbi.2014.09.003mailto:[email protected]:[email protected]://dx.doi.org/10.1016/j.cbi.2014.09.003http://-/?-http://-/?-http://-/?-http://-/?-http://crossmark.crossref.org/dialog/?doi=10.1016/j.cbi.2014.09.003&domain=pdfhttp://-/?-

8/10/2019 Astin B

2/9

toxicity-guided isolation method led to the identification of an

astin-rich fraction (71.2% of the relative content), from which a

halogenated cyclic pentapeptide astin B (Fig. 1A) was given [14].

Astin B is structurally similar to cyclochlorotine, a well-known

hepatotoxic mycotoxin isolated from Penicillium islandicum [15],

but the hepatotoxic effects and the underlying mechanisms wereunknown. In this study, we evaluated the effects of astin B on

the modulation of cell death, apoptosis and autophagy in human

normal liver L-02 cells. Our experiments indicate that astin B has

marked toxic effects in vitro and in vivo and induces hepatic cell

death mainly by apoptosis through a mitochondria/caspase-

dependent pathway. Astin B also induces autophagy in L-02 cells,

which appears to protect cells from apoptosis to some extent.

2. Materials and methods

2.1. Drugs and chemicals

Astin B (Fig. 1A) was obtained from previous experiments [14]

with a purity of more than 98%. Chlorogenic acid (CGA) wasobtained from Nanjing Zelang Medical Technology Company with

a purity of 98% (Nanjing, China). For experiments in cells, solutions

of astin B and CGA were prepared in DMSO and diluted to the

desired concentrations in FBS-free medium. The DMSO concentra-

tions in the experiments never exceeded 0.1%.

Fetal bovine serum (FBS) was obtained from Hyclone (Thermo,

South America). RPMI-1640 medium was obtained from Gibco

BRL (NY, USA.). Earles balanced salts solution (EBSS), 3-methylad-

enine (3-MA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra-

zolium bromide (MTT) were obtained from SigmaAldrich (St.

Louis, USA.). Antibodies for c-Jun N-terminal kinase (JNK), phos-

phorylated JNK (pJNK), Bax, Bcl-2, and caspase-3 were purchased

from Cell Signal Technology Inc. (Massachusetts, USA). Anti-LC3

antibody was obtained from MBL (Nagano, Japan). Anti-b-actinantibody and secondary anti-mouse and anti-rabbit antibodies

were obtained from Lianke Biotechnology (Hangzhou, China). All

of the other reagents were purchased from Amresco (Ohio, USA).

2.2. Cell culture

L-02 cells, a normal human liver cell line from the Cell Bank ofthe Chinese Academy of Sciences (Shanghai, China), were grown in

RPMI-1640 medium supplemented with 10% FBS, 100 U/mL peni-

cillin and 100 lg/mL streptomycin and incubated at 37C in ahumidified atmosphere (5% CO2). The medium was renewed every

2 days until the cells were grown to confluence.

2.3. Cytotoxicity assay

L-02 cells were seeded at an initial density of 1 105 cells/mL

in 96-well plates for 24 h and were incubated with fresh medium

containing different concentrations of astin B for 12, 24, and

48 h. After incubation, MTT was added to each well to reach a final

concentration of 0.5 mg/mL. The insoluble formazan was collected

and dissolved in DMSO and then measured using a microplatereader (Thermo, Finland) at a wavelength of 490 nm. For the assay

of lactate dehydrogenase (LDH), the cells were incubated with

astin B for 24 h. The supernatant was collected, and LDH activity

was determined with a commercial kit (Jiancheng, Nanjing, China)

in accordance with the manufacturers instructions.

2.4. Measurement of intracellular ROS and GSH

The generation of intracellular ROS was assessed using the

ROS-specific fluorescent dye 2,7-dichlorofluorescein diacetate

(DCFH-DA; Beyotime, Haimen, China). L-02 cells were seeded at

an initial density of 1 105 cells/mL in 96-well plates. After 12 h

exposure to astin B, the cells were washed with PBS, loaded with

10 lmol/L DCFH-DA at 37 C for 30 min away from light, rinsedthree times with serum-free culture media, and measured at an

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

0 15 30 45 60

Relativeviability

Astin B (M)

*

*

**

**

*

**

*

B

0

20

40

60

80

100

120

140

160

180

0 15 30 45 60

LDHrelease(%)

Astin B (M)

**

C

NH

NH

NHN

HN

O

O Cl

ClO

O

OH

O

OH

A

Fig. 1. Effects of astin B on cell viability and lactate dehydrogenase (LDH) activity in L-02 cells. (A) Chemical structure of astin B. (B) Cells were treated with astin B for 12, 24,

and 48 h, and cell viability was determined by the MTT assay. (C) Cells were treated with astin B for 24 h, and LDH leakage was measured using commercially available kits.

Data in B and C are expressed as the mean SD from three independent experiments with n= 6 for MTT and n= 4 for LDH. p< 0.05 compared with the control group (0 lM).

2 L. Wang et al. / Chemico-Biological Interactions 223 (2014) 19

8/10/2019 Astin B

3/9

excitation wavelength of 488 nm and an emission wavelength of

530 nm using a microplate fluorescence reader (MD, spectramax

M3, USA). For GSH assay, the cells were cultured with astin B for

24 h and then harvested by centrifuging. The intracellular GSH

level was determined with commercially available kits (Jiancheng,

Nanjing, China) in accordance with the manufacturers

instructions.

2.5. Analysis of mitochondrial membrane potential (DWm)

The membrane potential assay is based on the JC-1 dye: JC-1

emits green fluorescence when the DWm is relatively low. How-

ever, JC-1 aggregates and emits a red fluorescence when the

DWm is high [16]. The assay was performed with a JC-1 kit in

accordance with the manufacturers instructions (Beyotime).

Briefly, L-02 cells (1 105 cells/mL) in 6-well plates were sepa-

rately treated with or without astin B for 24 h and were incubated

with JC-1 staining solution (5 lg/mL) for 20 min at 37C in thedark. The cells were rinsed twice with JC-1 staining buffer and

were then observed and photographed using an Olympus

fluorescence microscope.

2.6. Measurement of cytochrome c (cyt c) content

After treatment with astin B for 24 h, the cells were lysed in

preparation buffer (250 mM sucrose, 20 mM HEPES-KOH (pH

7.4), 10 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA,

1 mM dithiothreitol, 2 lg/mL aprotinin and 1 mM phenylmethyl-sulfonyl fluoride) for 10 min on ice followed by centrifugation at

10,000 rpm for 20 min at 4 C to separate the cytosolic and mito-

chondrial fractions [17]. Protein concentrations were measured

with a bicinchoninic acid protein assay kit (Beyotime). The con-

tents of cyt c in the cytosol and mitochondria were determined

by ELISA kits (R&D Systems, Minnesota, USA) in accordance with

the manufacturers instructions.

2.7. Analysis of caspase activity

The activities of caspase-3 and caspase-9 were evaluated using

caspase-3 and caspase-9 activity assay kits (Beyotime). After a

24-h exposure, L-02 cells from 6-well plates were collected and

rinsed with cold PBS and later lysed using lysis buffer for 1 h on

ice. The cell lysates were centrifuged at 18,000 rpm for 10 min at

4C. The assays were performed in 96-well plates by incubating

10 lL of the cell lysate supernatant in 80lL reaction buffercontaining 10lL Ac-DEVD-pNA (for caspase-3) and 10lLAc-LEHD-pNA (for caspase-9). After further incubation at 37C

for 4 h, the absorbance was measured using a microplate reader

(Thermo, Finland) at a wavelength of 405 nm.

2.8. Flow cytometric analysis of apoptotic cells

2.8.1. Sub-G1 peak

The apoptosis induced by astin B was determined using a cell

cycle and apoptosis analysis kit (Beyotime) in accordance with

the manufacturers instructions. In brief, approximately 1 105

L-02 cells were cultured in 6-well plates and separately treated

with or without astin B for 24 h. After treatment, the cells were col-

lected and fixed with 70% ethanol at 4 C overnight, centrifuged at

800 rpm for 5 min and stained with 500 lL of buffer, 25 lL of pro-pidium iodide (PI), and 10 lL of RNase A in the dark for 30 min at37C. Next, the cells were analyzed using a flow cytometer (Becton

Dickinson, New Jersey, USA). The apoptotic cells with hypodiploid

DNA content were measured by quantifying the sub-G1 peak in the

cell cycle pattern. For each experiment, 10,000 events wererecorded per sample.

2.8.2. Annexin-V/PI staining

After 24 h treatment, the L-02 cells were gently trypsinized and

washed once with PBS, centrifuged at 800 rpm for 5 min, and

resuspended in 200 lL of binding buffer. After gentle pipetting,the cells were stained by 3 lL of Annexin-V-FITC (AV) and 3 lLof propidium iodide (PI) (BD Pharmingen, California, USA) for

15 min at room temperature in the dark and analyzed using a flow

cytometer (Becton Dickinson, New Jersey, USA). For each experi-ment, 10,000 events were recorded per sample.

2.9. Acridine orange (AO) staining of autophagic cells

Autophagic cells were detected by AO staining in accordance

with published procedures [18]. In brief, approximately 1 105

cells were seeded in 24-well plates and incubated with astin B

for 12 h, washed with PBS, and colored with AO (1lg/mL)(Generay, Shanghai, China) at 37C for 1 min in the dark. The

cells were then observed and photographed by fluorescence

microscopy.

2.10. Western blot analysis

The L-02 cells (approximately 6 105 in number) were

extracted with 100lL of lysate buffer, which consists of 1 MTrisHCl, 50% glycerine, 1.0% bromophenol blue, 10% SDS, andb-mercaptoethanol. After boiling for 10 min, the extracted samples

were loaded at 20 lL/per lane, fractionated on 1215% trisglycineprecast gels, and then transferred to a PVDF membrane (Millipore,

Billerica, USA). The proteins were probed with primary antibodies

and HRP-labeled secondary antibodies and visualized using super

ECL detection reagent (Beyotime).

2.11. Animal experiments

2.11.1. Animals

Male ICR mice (68 weeks of age) were obtained from theLaboratory Animal Center of Nanjing Qinglongshan (Nanjing,

China). They were maintained with free access to pellet food and

water in plastic cages at 21 2 C and kept on a 12-h light/dark

cycle. The care and treatment of these mice were in accordance

with the Provisions and General Recommendations of the Chinese

Experimental Animals Administration Legislation. This study was

approved by the Animal Ethics Committee of the School of Chinese

Materia Medica, China Pharmaceutical University.

2.11.2. Hepatotoxicity in mice

All of the mice were acclimatized for 3 days prior to experimen-

tal procedures. Two groups of mice (10 per group) were adminis-

tered daily with astin B (10 mg/kg, suspended in distilled water)

or distilled water by gavage for 7 days. One hour after the lastadministration, the mice were killed by cervical dislocation after

isoflurane inhalation, blood was collected from the orbital sinus

and centrifuged at 12,000 rpm for 5 min at 4C to obtain serum

for the test of alanine aminotransferase (ALT) and aspartate amino-

transferase (AST) activities by commercial kits (Jiancheng, Nanjing,

China). Liver tissue was excised and fixed in 10% phosphate-

buffered formalin and embedded in paraffin. Sections were

prepared and stained with hematoxylin and eosin (H&E) for histo-

pathological analysis.

2.12. Statistical analysis

The data are expressed as the means SD (standard deviation)

from at least three independent experiments. Statistical analyseswere performed using one-way analysis of variance (ANOVA)

L. Wang et al./ Chemico-Biological Interactions 223 (2014) 19 3
http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-

8/10/2019 Astin B

4/9

followed by Students two-tailed t-test. Values of p< 0.05 were

considered to be statistically significant.

3. Results

3.1. Astin B inhabits proliferation of L-02 cells

The potential cytotoxic effect of astin B was investigated byincubating L-02 cells with astin B at different concentrations for

12, 24, and 48 h. The results of the MTT assay showed that astin

B decreased cell viability in a concentration-dependent and time-

dependent manner (Fig. 1B). After 48 h treatment, the cell viability

decreased to 39.22% at 60 lM. Leakage of LDH from cells is an indi-cator of cell necrosis. As shown inFig. 1C, there was no significant

leakage of LDH in cells after treatment with astin B at 15 and

30 lM for 24 h. Increased LDH leakage was observed only in cellsexposed to high concentrations of 45 and 60 lM.

3.2. Astin B induces oxidative stress

To determine whether astin B induces oxidative stress, we

observed the effects of astin B on ROS production and GSH levels.Compared with untreated cells, the intracellular ROS levels

increased significantly when the cells were exposed to astin B for

12 h (Fig. 2A). In contrast, the GSH levels in the cells were reduced

in a concentration-dependent manner after treatment with astin B

for 24 h (Fig. 2B). Astin B promotes ROS production and decreases

GSH levels. Because JNK activity and oxidative stress are tightly

associated, we further observed the effects of astin B on JNK phos-

phorylation in cells treated with astin B at 30 lM for 0, 3, 6, 12, 24,and 48 h. As shown inFig. 2C, the phosphorylation of JNK was obvi-

ously enhanced after treatment with astin B for 3, 6, and 12 h.

Chlorogenic acid (CGA) is a well-known antioxidant and potential

hepatoprotective [19]. After pretreatment with CGA for 12 h, the

cell viability of astin B-treated (15, 30, and 60lM) groupsincreased by 7.76% (p< 0.001), 9.24% (p< 0.01), and 6.03%

(p< 0.05), compared with the corresponding groups without pre-

treated by CGA (Fig. 2D). This may be evidenced from the reverse

side that astin B can cause oxidative stress and liver injury.

3.3. Astin B induces mitochondrial dysfunction

To assess whether astin B affects the functioning of mitochon-

dria, we determined the changes in the mitochondrial membrane

potential (DWm). The results showed that astin B induced concen-

tration-dependent DWm collapse in L-02 cells after incubation

with astin B for 24 h (Fig. 3A). Furthermore, the release of cyt c

from the mitochondria into the cytosol in the L-02 cells was also

detected. Exposure of cells to astin B for 24 h significantly

increased the cyt c content of the cytosol fraction (Fig. 3B).

3.4. Astin B regulates Bcl-2/Bax expression and increases caspase-3

and -9 activity

As apoptotic proteins, Bcl-2 is a powerful antagonist of apopto-tic death programs, whereas Bax accelerates apoptotic death and

counters the death repressor activity of Bcl-2. The ratio of Bcl-2

to Bax determines the survival or death of the cells following an

apoptotic stimulus[20]. Treatment of L-02 cells with astin B signif-

icantly induced the expression of pro-apoptotic Bax and decreased

the expression of anti-apoptotic Bcl-2 in a concentration-

dependent fashion (Fig. 4A). Thus, astin B significantly increased

the Bax/Bcl-2 ratio (Fig. 4B) in a concentration-dependent manner,

leading to a state associated with apoptosis. Caspases are most

likely the most important effector molecules for the execution of

apoptosis[21]. When the L-02 cells were treated with astin B for

0

50

100

150

200

250

300

0 15 30 45 60

GSH(nmol/mgprotein)

Astin B (M)

*

**

*

B

0

1000

2000

3000

4000

5000

0 15 30 60

DCFfluorescenceintensity

Astin B (M)

A C* * *

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 15 30 60

Relativeviability

Astin B (M)

D

Fig. 2. Astin B induced oxidative stress in L-02 cells. (A) Cells were treated with astin B for 12 h, and intracellular ROS was assessed by the ROS-specific fluorescent dye DCFH-

DA. (B) Cells were treated with astin B for 24 h, and the intracellular GSH level was determined using commercially available kits. (C) Cells were treated with astin B (30 lM)at regular intervals, and phosphorylated JNK was determined using Western blot analysis. (D) Cells were pretreated with chlorogenic acid (CGA+, 100 lM) for 12 h, followingtreated with astin B for 24 h, and cell viability was determined by the MTT assay. Data in (A, B, and D) are expressed as the mean SD from three independent experiments

withn = 46.

p< 0.05 compared with the control group (0 lM), #p< 0.05 compared with the corresponding CGA group. The results shown in C are representative of threeindependent experiments.


8/10/2019 Astin B

5/9

0

50

100

150

200

250

300

350

400

Cytc(ng/m

gprotein)

Astin B (M)

0 15 30 45 60 0 15 30 45 60

Cytosol Mitochondria

*

*

* *

*

*

BA

Fig. 3. Astin B decreased the mitochondrial membrane potential (DWm) and promoted cytochrome c release. (A) L-02 cells were treated with astin B for 24 h and

subsequently incubated with JC-1 dye for 20 min, then observed and photographed by an Olympus fluorescence microscope. The proportion of green fluorescence emission

represents the DWm collapse degree. (B) Cells were treated with astin B for 24 h, and the content of cytochrome c was measured with ELISA kits. The results shown in A are

representative of three independent experiments. Data in (B) are expressed as the mean SD from three independent experiments with n = 4. p< 0.05 compared with the

control group (0 lM). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

0

1

2

3

4

5

6

7

8

9

0 15 30 45 60

Foldofcontrol

Astin B (M)

* *

*

*D

Caspase-3

0

1

2

3

4

0 15 30 45 60

Foldofcontrol

Astin B (M)

*

*

**

C Caspase-9

0

1

2

3

4

5

0 15 30 45 60

RatioofBax/Bcl-2

Astin B (M)

*

*

*

BA

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 15 30 60

Relativeviability

Astin B (M)

zVAD - zVAD +

**

*

E

Fig. 4. Astin B regulated Bcl-1/Bax expression and increased activities of caspases-9 and -3 in L-02 cells. Cells were treated with astin B for 24 h: (A) Bax and Bcl-2 expressions

were determined by Western blot, and (B) the ratio of Bax/Bcl-2 was calculated by densitometry. Enzymatic activities of (C) caspase-9 and (D) caspase-3 were measured using

commercial kits. (E) Cell viability in the presence of z-VAD-fmk (z-VAD+, 50 lM), a pan-caspase inhibitor, was determined by the MTT assay. The results shown in (A) are

representative of three independent experiments. The data in B-E are expressed as the mean SD from three independent experiments with n = 3.

p< 0.05 compared withthe control group (0 lM) in (BD) or with the corresponding z-VAD-group in E.

http://-/?-http://-/?-

8/10/2019 Astin B

6/9

24 h, the activities of caspases-3 and -9 were increased in concen-

tration-dependent manners (Fig. 4C and D). Co-treatment of astin B

with z-VAD-fmk (a pan-caspase inhibitor) could increase the via-

bility of the L-02 cells to some extent, compared with the groups

that were only treated by astin B with corresponding concentra-

tions (Fig. 4E). These results suggest that astin B most likely

mediates apoptotic cell death and that they do so mainly in a

caspase-dependent manner.

3.5. Astin B induces apoptotic cell death

To confirm whether astin B induced cell death through apopto-

sis, we analyzed the sub-G1 peaks, i.e., the population of apoptotic

cells, of the L-02 cells. After treatment of the cells with astin B at 0,

15, 30, 45, and 60 lM for 24 h, the percentage of the sub-G1 peakgradually increased in a concentration-dependent manner from

4.09% to 14.22% (Fig. 5A and B), indicating apoptotic cell death

induced by astin B in L-02 cells. This finding was further evidenced

by the Annexin-V/PI staining method, which can clearly discrimi-

nate among the four types of cells, i.e., unaffected cells (AV/

PI), early apoptotic cells (AV+/PI), early necrotic and late apop-

totic cells (AV+/PI+), and late necrotic cells (died without apopto-

sis) (AV/PI+). After treatment with astin B at same

concentrations for 24 h, the early apoptotic L-02 cells increased

obviously in a concentration-dependent fashion from 8.03% to

47.12%, along with lower increases of necrotic and late apoptotic

cells from 4.64% to 16.33% (Fig. 5C and D). These results indicate

that apoptosis is one of the major ways for astin B to induce L-02

cell death, which may be one of the main hepatotoxic components

in RAT.

3.6. Astin B induces autophagy against apoptosis

Autophagy is important for cell death decisions and can protect

cells by preventing them from undergoing apoptosis [22]. There-

fore, a method of AO staining was employed to detect autophagiceffects in L-02 cells. As shown in Fig. 6A, after exposure to astin

B for 12 h, autophagic vacuoles were visible in the cells. Further-

more, the expressions of LC3-I/LC3-II and p62, both key indicators

for autophagosome formation[23,24],were examined by Western

blot analysis. The results showed that the treatment of L-02 cells

with astin B significantly enhanced LC3-II and reduced p62 expres-

sion (Fig. 6B). These data indicate that the stimulus of astin B

induces both autophagy and apoptosis in hepatocytes. In addition,

the connection between autophagy and apoptosis was investigated

with astin B-induced L-02 cells by co-treatment with 3-MA (an

autophagy inhibitor) or z-VAD-fmk (a caspase inhibitor).

Compared with treatment with astin B alone, co-treatment with

3-MA obviously attenuated LC3-II expression, rapidly enhanced

caspase-3 expression, and significantly reduced cell viability in

L-02 cells (Fig. 6C and D). On the other hand, co-treatment with

z-VAD-fmk resulted in an accrual of autophagic pathway evi-

denced by increased LC3-II and decreased p62 (Fig. 6E), which

may partially explain why the cell viability was only increased

slightly on blocking apoptosis (Fig. 4E). These results indicated that

astin B-induced LC3-II enhancement and p62 redaction were the

0

2

4

6

8

10

12

14

16

0 15 30 45 60

Cellsofsub-G1peak(%)

Astin B (M)

*

*

**

B

A

C

0

10

20

30

40

50

60

0 15 30 45 60

Cellnumber(%)

Astin B (M)

AV+ PI- A V+PI+

* * *

*

*

*

*

*

D

Fig. 5. Astin B increased the apoptotic L-02 cells. Cells were treated with astin B for 24 h, and apoptotic cells were determined by flow cytometry. (A and B) The population of

sub-G1 peaks (indicated by an arrow) was measured by propidium iodide staining, and (C and D) apoptotic and necrotic cells were clarified by Annexin V-FITC (AV) and

propidium iodide (PI) staining. The results shown in (A) and (C) are representative of three independent experiments. The data in (B) and (D) are expressed as the mean SDfrom three independent experiments. p< 0.05 compared with the control group (0 lM).


8/10/2019 Astin B

7/9

0

0.2

0.4

0.6

0.8

1

1.2

Relativeviability

P 0.05

DC

E

B

A

Fig. 6. Astin B induced autophagy in L-02 cells to protect from apoptosis. (A) After treatment with astin B (30lM) for 12 h, autophagy in the L-02 cells was detected byacridine orange (AO) staining, and orange-colored vacuoles indicated positive results, Earles balanced salt solution (EBSS) as positive control. (B) After treatment with astin B

(15, 30, 60 lM) for 12 h, the levels of p62 and LC3-I/LC3-II in L-02 cells were determined by Western blot analysis. (C and D) L-02 cells were exposed to astin B (60 lM) with orwithout the presence of the autophagy inhibitor 3-MA (2 mM) for 12 h, the levels of LC3-I/LC3-II and procaspase-3/cleaved caspase-3 were determined by Western blot

analysis, and cell viability was measured by the MTT assay. (E) L-02 cells were treated with astin B (60 lM) with or without the presence of the pan-caspase inhibitor z-VAD-fmk (50 lM) for 12 h, the levels of p62 and LC3-I/LC3-II were determined by Western blot analysis. The results shown in A are representative of three independentexperiments. The results shown in (B, C, and E) are representative of three independent experiments. The data in (D) are expressed as the mean SD from three independent

experiments.

**

**

0

20

40

60

80

100

120

140

160

Control Astin B

Enzymeactivity(U/L)

ALT

AST

A B

Fig. 7. Hepatotoxicity of astin B in mice. Ten mice were orally administered with vehicle (control group) or astin B at 10 mg/kg once daily for 7 consecutive days. (A) ALT and

AST levels in serum were determined by commercial kits. (B) Slices of liver were stained with H&E for histopathological analysis. The data in (A) are expressed as themean SD, p< 0.01 compared with the control group; the results shown in (B) are representative of five independent experiments at a magnification of 200 .

http://-/?-http://-/?-

8/10/2019 Astin B

8/9

real events associated with autophagosomes and that astin B-

induced apoptotic cell death will be accelerated in the absence of

autophagy in hepatocytes.

3.7. Astin B induces liver injury in mice

To investigate whether astin B induces liver injury in vivo, we

observed the effects of orally administered astin B on the liver cells

in mice. Compared to the control group, the serum levels of ALT

and AST in mice treated with astin B were elevated about 11.9

and 2.26 times, respectively (Fig. 7A). The liver injury of astin B

was further confirmed by histopathological examination, as

evidenced by diffuse hydropic degeneration of hepatocytes with

moderate inflammatory cell infiltration (Fig. 7B). The results

indicated that, consistent with its action in vitro, astin B could also

induce liver injuryin vivo.

4. Discussion

Our previous work suggested that pentapeptides, especially the

cyclic ones, were most likely the principal toxic components in RAT

[14]. Astins are a class of halogenated cyclic pentapeptides con-

tained in RAT. Although astins have been reported to kill tumor

cells and prevent colitis through regulation of apoptosis [1113],

our results clearly showed that astin B, one of the cyclic pentapep-

tides, induced hepatic cell death mainly by ROS-associated apopto-

sis via a mitochondria-dependent pathway. Such cell death will

lead to liver injury, which was evidenced by thein vivoexperiment.

On the other hand, astin B also positively regulated autophagy, and

this action partially attenuated apoptotic cell death.

MTT assays showed that astin B effectively inhibited cell prolif-

eration and survival. The inhibitory percentage reached 60.78% at

60 lM after 48 h exposure. Apoptosis and necrosis are the mostfrequent events in xenobiotic metabolism-induced liver injury

[25]. Astin B substantially increased cell death, while the leakageof LDH, an indicator of necrosis, only moderately increased in cells

that were exposed to high concentrations. This finding indicates

that apoptosis is most likely the major event that is responsible

for the cell death.

Uncontrolled ROS production and/or decreased antioxidant

defenses result in oxidative stress, which has been considered to

be an important pathogenic element for the initiation of hepato-

toxicity[4,6]. GSH is a major antioxidant that guards cells against

oxidative injury by diminishing ROS. Therefore alterations in the

GSH level can be monitored as an indication of oxidative stress

in cells [26]. Astin B treatment enhanced ROS production with

downregulation of GSH levels in L-02 cells, indicating that oxida-

tive stress occurred following the astin B stimulus. Changes in

ROS and GSH act as intracellular signals and can evoke inflamma-tion by the regulation of transcription factor molecules through

JNK activation [27]. Consistent with this view, we observed

enhanced pJNK expression when the cells were exposed to astin

B. Moreover, the inhibited cell proliferation and survival by astin

B could be reversed by antioxidants, such as CGA, to some extent.

All these alterations indicate that the astin B stimulus induces

ROS-associated inflammation in hepatocytes.

Given that the mitochondria are the major intracellular source

of ROS, we wondered whether ROS-associated inflammation would

impair mitochondrial function. Our experiments showed that astin

B induces the collapse ofDWm with an increased release of cyt c

from the mitochondria into the cytosol. This result suggests that

mitochondrial dysfunction results in the opening of the mitochon-

drial permeability transition pores, leading to the release of cyt cfrom the mitochondria.

In view of the link between mitochondrial dysfunction and

apoptosis, the participation of the Bcl-2 family (which contains

both pro- and anti-apoptotic proteins) is a key event because of

the regulative effects of these proteins on mitochondrial dysfunc-

tion during apoptosis. In such regulation, Bcl-2 is an anti-apoptotic

member, while Bax is a pro-apoptotic multidomain effector protein

[28]. Increased pro-apoptotic proteins can actively permeabilize

the outer mitochondrial membrane and promote the release ofintermembrane space proteins, such as cyt c, into the cytosol. Cyt

c is required for the activation of caspase-9. Generally, activated

caspase-9 is involved in the mitochondrial pathway, and it acti-

vates downstream caspase-3, thereby triggering cell apoptosis

[29]. The increased ratio of Bax/Bcl-2 observed in cells treated with

astin B indicates that astin B induces mitochondrial outer mem-

brane permeabilization, which discharges cyt c into the cytosol.

Furthermore, the increased activities of caspases-9 and -3 observed

in treated cells suggest that the astin B-induced apoptosis may fol-

low a caspase-dependent intrinsic mitochondrial pathway. The ini-

tiation of apoptosis induced by astin B was further characterized

by an increased sub-G1 peak population during the cell cycle and

a predominant population of early apoptotic cells among the dead

cells.

Autophagy functions as a cytoplasmic quality control mecha-

nism to remove protein aggregates and damaged organelles.

Although autophagy has been observed in many dying cells, it is

generally accepted that autophagy is a pro-survival and protective

pathway [30,31]. Apoptosis and autophagy are genetically regu-

lated processes that regulate cell fate. The functional relationship

between apoptosis and autophagy is quite complex. In several sce-

narios, autophagy prevents cell death and de facto suppresses

apoptosis; and it also appears that similar stimuli can induce either

apoptosis or autophagy [32]. AO-stained autophagic vacuoles,

enhanced LC3-II and reduced p62 in L-02 cells treated with astin

B confirmed that astin B can also induce autophagy in hepatocytes.

After co-treatment with the autophagy inhibitor 3-MA, decreased

cell viability and dramatically increased expression of cleaved

caspase-3 were observed. Conversely, co-treatment with caspaseinhibitor z-VAD-fmk led to an enhanced autophagy for astin

B-treated cells evidenced by increased LC3-II and decreased p62

expression. These results indicate that apoptosis and autophagy

induced by astin B were interacted and that autophagy could

effectively protect the cells from B-induced cell apoptosis. The

protective pathway of autophagy,i.e., whether autophagy is a sec-

ondary result of cell death that functions to remove damaged

organelles or if it is directly induced by astin B in an apoptosis-

independent manner, remains to be determined.

In summary, our studies suggest that astin B will provoke oxi-

dative stress-associated inflammation and induce hepatocyte

apoptosis through a mitochondria-dependent pathway, leading to

liver injury. However, astin B will also induce autophagy to protect

cells from apoptosis and to alleviate hepatic injury to some extent.Furthermore, we found that an astin-rich fraction (astins were

71.2% of the relative content)[14] induces apoptosis in the same

fashion (data not shown). As a result, astins (including astin B)

can be considered to be the major hepatotoxic substances in the

herbal medicine derived from A. tataricus. Owing to their special

and interesting structures, previous studies of astins focused on

their biological activities, and their toxic or side effects were often

overlooked. We hope that our finding will be helpful for providing

advice regarding drug safety for the clinical use of RAT and the

development of new pharmaceuticals.

Conflict of Interest

The authors declare that there are no conflicts of interest.


8/10/2019 Astin B

9/9

Transparency Document

TheTransparency documentassociated with this article can be

found in the online version.

Acknowledgments

This work was financially supported by the National Natural

Science Foundation of China (30772702) and the National New

Drug Innovation Major Project of China (2011ZX09307-002-02).

References

[1] F. Stickel, E. Patsenker, D. Schuppan, Herbal hepatotoxicity, J. Hepatol. 43(2005) 901910.

[2] H. Malhi, G.J. Gores, J.J. Lemasters, Apoptosis and necrosis in the liver: a tale oftwo deaths?, Hepatology 43 (2006) S31S44

[3] G. Kroemer, L. Galluzzi, P. Vandenabeele, J. Abrams, E.S. Alnemri, E.H.Baehrecke, M.V. Blagosklonny, W.S. Eldeiry, P. Golstein, D.R. Green, M.Hengartner, R.A. Knight, S. Kumar, S.A. Lipton, W. Malorni, G. Nunez, M.E.Peter, J. Tschopp, J. Yuan, M. Piacentini, B. Zhivotovsky, G. Melino, Classificationof cell death: recommendations of the Nomenclature Committee on cell death,Cell Death Differ. 16 (2009) 311.

[4] J.L. Martindale, N.J. Holbrook, Cellular response to oxidative stress: signalingfor suicide and survival, J. Cell Physiol. 192 (2002) 115.

[5] R. Heidari, H. Babaei, M. Eghbal, Mechanisms of methimazole cytotoxicity inisolated rat hepatocytes, Drug Chem. Toxicol. 36 (2013) 403411.

[6] D. Pessayre, B. Fromenty, A. Berson, M.A. Robin, P. Lettron, R. Moreau, A.Mansouri, Central role of mitochondria in drug-induced liver injury, DrugMetab. Rev. 44 (2012) 3487.

[7] M. Giorgio, E. Migliaccio, F. Orsini, D. Paolucci, M. Moroni, C. Contursi, G.Pelliccia, L. Luzi, S. Minucci, M. Marcaccio, P. Pinton, R. Rizzuto, P. Bernardi, F.Paolucci, P.G. Pelicci, Electron transfer between cytochrome c and p66 (Shc)generates reactive oxygen species that trigger mitochondrial apoptosis, Cell122 (2005) 221233.

[8] S. Lim, S.J. Lee, K.W. Nam, K.H. Kim, W. Mar, Hepatoprotective effects ofreynosin against thioacetamide-induced apoptosis in primary hepatocytes andmouse liver, Arch. Pharm. Res. 36 (2013) 485494.

[9] D. Han, L. Dara, S. Win, T.A. Than, L. Yuan, S.Q. Abbasi, Z.X. Liu, N. Kaplowitz,Regulation of drug-induced liver injury by signal transduction pathways:critical role of mitochondria, Trends Pharmacol. Sci. 34 (2013) 243253.

[10] C. Bincoletto, A. Bechara, G.J. Pereira, C.P. Santos, F. Antunes, J. Peixoto da-Silva,M. Muler, R.D. Gigli, P.T. Monteforte, H. Hirata, A. Jurkiewicz, S.S. Smaili,Interplay between apoptosis and autophagy, a challenging puzzle: newperspectives on antitumor chemotherapies, Chem. Biol. Interact. 206 (2013)279288.

[11] H. Morita, S. Nagashima, K. Takeya, H. Itokawa, Y. Iitaka, Structures andconformation of antitumour cyclic pentapeptides, astins A, B and C, from Astertataricus, Tetrahedron 51 (1995) 11211132.

[12] R. Cozzolino, P. Palladino, F. Rossi, G. Cali, E. Benedetti, P. Laccetti,Antineoplastic cyclic astin analogues kill tumour cellsvia caspase-mediatedinduction of apoptosis, Carcinogenesis 26 (2005) 733739.

[13] Y. Shen, Q. Luo, H.M. Xu, F.Y. Gong, X.B. Zhou, Y. Sun, X.F. Wu, W. Liu, G.Z. Zeng,N.H. Tan, Q. Xu, Mitochondria-dependent apoptosis of activated Tlymphocytes induced by astin C, a plant cyclopeptide, for preventing murineexperimental colitis, Biochem. Pharmacol. 82 (2011) 260268.

[14] X.D. Liu, P.P. Cao, C.F. Zhang, X.H. Xu, M. Zhang, Screening and analyzingpotential hepatotoxic compounds in the ethanol extract of Asteris Radix byHPLC/DAD/ESI-MSn technique, J. Pharm. Biomed. Anal. 6768 (2012) 5162.

[15] K. Terao, E. Ito, T. Tatsuno, Liver injuries induced by cyclochlorotine isolatedfromPenicillium islandicum, Arch. Toxicol. 55 (1984) 3946.

[16] A. Cossarizza, M. Baccaranicontri, G. Kalashnikova, C. Franceschi, A new

method for the cytofluorometric analysis of mitochondrial membranepotential using the J-aggregate forming lipophilic cation 5,50 ,6,60-tetrachloro-1,10 ,3,30-tetraethylbenzimidazolcarbocyanine iodide (JC-1),Biochem. Biophys. Res. Commun. 197 (1993) 4045.

[17] V.A. Tyurin, Y.Y. Tyurina, W. Feng, A. Mnuskin, J.M. Jiang, Tang, X. Zhang, Q.Zhao, P.M. Kochanek, R.S. Clark, H. Bayir, V.E. Kagan, Mass-spectrometriccharacterization of phospholipids and their primary peroxidation products inrat cortical neurons during staurosporine-induced apoptosis, J. Neurochem.107 (2008) 16141633.

[18] S. Paglin, T. Hollister, T. Delohery, N. Hackett, M. McMahill, E. Sphicas, D.Domingo, J. Yahalom, A novel response of cancer cells to radiation involvesautophagy and formation of acidic vesicles, Cancer Res. 61 (2001) 439444.

[19] A. Kapil, I.B. Koul, O.P. Suri, Antihepatotoxic effects of chlorogenic acid fromAnthocephalus cadamba, Phytother. Res. 9 (1995) 189193.

[20] Z.N. Oltval, C.L. Milliman, S.J. Korsmeyer, Bcl-2 heterodimerizes in vivo with aconserved homolog, Bax, that accelerates programmed cell death, Cell 74(1993) 609619.

[21] M. Boyce, A. Degterev, J. Yuan, Caspases: an ancient cellular sword ofDamocles, Cell Death Differ. 11 (2004) 2937.

[22] A. Thorburn, Apoptosis and autophagy: regulatory connections between twosupposedly different processes, Apoptosis 13 (2008) 19.

[23] Y. Kouroku, E. Fujita, I. Tanida, T. Ueno, A. Isoai, H. Kumagai, S. Ogawa, R.J.Kaufman, E. Kominami, T. Momoi, ER stress (PERK/eIF2 alpha phosphorylation)mediates the polyglutamine-induced LC3 conversion, an essential step forautophagy formation, Cell Death Differ. 14 (2007) 230239.

[24] E. Itakura, N. Mizushima, P62 targeting to the autophagosome formation siterequires self-oligomerization but not LC3 binding, J. Cell Biol. 192 (2011) 1727.

[25] H. Malhi, M.E. Guicciardi, G.J. Gores, Hepatocyte death: a clear and presentdanger, Physiol. Rev. 90 (2010) 11651194.

[26] L. Yuan, N. Kaplowitz, Glutathione in liver diseases and hepatotoxicity, Mol.Aspects Med. 30 (2009) 2941.

[27] S.J. Lee, M.S. Kim, J.Y. Park, J.S. Woo, Y.K. Kim, 15-Deoxy-delta (12,14)-prostaglandin J (2) induces apoptosis via JNK-mediated mitochondrialpathway in osteoblastic cells, Toxicology 248 (2008) 121129 .

[28] R.J. Youle, A. Strasser, The Bcl-2 protein family: opposing activities thatmediate cell death, Nat. Rev. Mol. Cell Biol. 9 (2008) 4759.

[29] N.N. Danial, S.J. Korsmeyer, Cell death: critical control points, Cell 116 (2004)205219.

[30] P. Boya, R.A. Gonzalez-Polo, N. Casares, J.L. Perfettini, P. Dessen, N. Larochette,D. Metivier, D. Meley, S. Souquere, T. Yoshimori, G. Pierron, P. Codogno, G.Kroemer, Inhibition of macroautophagy triggers apoptosis, Mol. Cell Biol. 25(2005) 10251040.

[31] N. Mizushima, B. Levine, A.M. Cuervo, D.J. Klionsky, Autophagy fights diseasethrough cellular self digestion, Nature 451 (2008) 10691075.

[32] M.C. Maiuri, E. Zalckvar, A. Kimchi, G. Kroemer, Self-eating and self-killing:crosstalk between autophagy and apoptosis, Nat. Rev. Mol. Cell Biol. 8 (2007)741752.

http://dx.doi.org/10.1016/j.cbi.2014.09.003http://refhub.elsevier.com/S0009-2797(14)00251-8/h0005http://refhub.elsevier.com/S0009-2797(14)00251-8/h0005http://refhub.elsevier.com/S0009-2797(14)00251-8/h0005http://refhub.elsevier.com/S0009-2797(14)00251-8/h0010http://refhub.elsevier.com/S0009-2797(14)00251-8/h0010http://refhub.elsevier.com/S0009-2797(14)00251-8/h0015http://refhub.elsevier.com/S0009-2797(14)00251-8/h0015http://refhub.elsevier.com/S0009-2797(14)00251-8/h0015http://refhub.elsevier.com/S0009-2797(14)00251-8/h0015http://refhub.elsevier.com/S0009-2797(14)00251-8/h0015http://refhub.elsevier.com/S0009-2797(14)00251-8/h0015http://refhub.elsevier.com/S0009-2797(14)00251-8/h0020http://refhub.elsevier.com/S0009-2797(14)00251-8/h0020http://refhub.elsevier.com/S0009-2797(14)00251-8/h0020http://refhub.elsevier.com/S0009-2797(14)00251-8/h0025http://refhub.elsevier.com/S0009-2797(14)00251-8/h0025http://refhub.elsevier.com/S0009-2797(14)00251-8/h0025http://refhub.elsevier.com/S0009-2797(14)00251-8/h0030http://refhub.elsevier.com/S0009-2797(14)00251-8/h0030http://refhub.elsevier.com/S0009-2797(14)00251-8/h0030http://refhub.elsevier.com/S0009-2797(14)00251-8/h0030http://refhub.elsevier.com/S0009-2797(14)00251-8/h0035http://refhub.elsevier.com/S0009-2797(14)00251-8/h0035http://refhub.elsevier.com/S0009-2797(14)00251-8/h0035http://refhub.elsevier.com/S0009-2797(14)00251-8/h0035http://refhub.elsevier.com/S0009-2797(14)00251-8/h0035http://refhub.elsevier.com/S0009-2797(14)00251-8/h0040http://refhub.elsevier.com/S0009-2797(14)00251-8/h0040http://refhub.elsevier.com/S0009-2797(14)00251-8/h0040http://refhub.elsevier.com/S0009-2797(14)00251-8/h0045http://refhub.elsevier.com/S0009-2797(14)00251-8/h0045http://refhub.elsevier.com/S0009-2797(14)00251-8/h0045http://refhub.elsevier.com/S0009-2797(14)00251-8/h0050http://refhub.elsevier.com/S0009-2797(14)00251-8/h0050http://refhub.elsevier.com/S0009-2797(14)00251-8/h0050http://refhub.elsevier.com/S0009-2797(14)00251-8/h0050http://refhub.elsevier.com/S0009-2797(14)00251-8/h0050http://refhub.elsevier.com/S0009-2797(14)00251-8/h0055http://refhub.elsevier.com/S0009-2797(14)00251-8/h0055http://refhub.elsevier.com/S0009-2797(14)00251-8/h0055http://refhub.elsevier.com/S0009-2797(14)00251-8/h0055http://refhub.elsevier.com/S0009-2797(14)00251-8/h0055http://refhub.elsevier.com/S0009-2797(14)00251-8/h0060http://refhub.elsevier.com/S0009-2797(14)00251-8/h0060http://refhub.elsevier.com/S0009-2797(14)00251-8/h0060http://refhub.elsevier.com/S0009-2797(14)00251-8/h0060http://refhub.elsevier.com/S0009-2797(14)00251-8/h0060http://refhub.elsevier.com/S0009-2797(14)00251-8/h0065http://refhub.elsevier.com/S0009-2797(14)00251-8/h0065http://refhub.elsevier.com/S0009-2797(14)00251-8/h0065http://refhub.elsevier.com/S0009-2797(14)00251-8/h0065http://refhub.elsevier.com/S0009-2797(14)00251-8/h0070http://refhub.elsevier.com/S0009-2797(14)00251-8/h0070http://refhub.elsevier.com/S0009-2797(14)00251-8/h0070http://refhub.elsevier.com/S0009-2797(14)00251-8/h0070http://refhub.elsevier.com/S0009-2797(14)00251-8/h0075http://refhub.elsevier.com/S0009-2797(14)00251-8/h0075http://refhub.elsevier.com/S0009-2797(14)00251-8/h0075http://refhub.elsevier.com/S0009-2797(14)00251-8/h0075http://refhub.elsevier.com/S0009-2797(14)00251-8/h0080http://refhub.elsevier.com/S0009-2797(14)00251-8/h0080http://refhub.elsevier.com/S0009-2797(14)00251-8/h0080http://refhub.elsevier.com/S0009-2797(14)00251-8/h0080http://refhub.elsevier.com/S0009-2797(14)00251-8/h0080http://refhub.elsevier.com/S0009-2797(14)00251-8/h0080http://refhub.elsevier.com/S0009-2797(14)00251-8/h0080http://refhub.elsevier.com/S0009-2797(14)00251-8/h0080http://refhub.elsevier.com/S0009-2797(14)00251-8/h0080http://refhub.elsevier.com/S0009-2797(14)00251-8/h0080http://refhub.elsevier.com/S0009-2797(14)00251-8/h0080http://refhub.elsevier.com/S0009-2797(14)00251-8/h0085http://refhub.elsevier.com/S0009-2797(14)00251-8/h0085http://refhub.elsevier.com/S0009-2797(14)00251-8/h0085http://refhub.elsevier.com/S0009-2797(14)00251-8/h0085http://refhub.elsevier.com/S0009-2797(14)00251-8/h0085http://refhub.elsevier.com/S0009-2797(14)00251-8/h0085http://refhub.elsevier.com/S0009-2797(14)00251-8/h0090http://refhub.elsevier.com/S0009-2797(14)00251-8/h0090http://refhub.elsevier.com/S0009-2797(14)00251-8/h0090http://refhub.elsevier.com/S0009-2797(14)00251-8/h0095http://refhub.elsevier.com/S0009-2797(14)00251-8/h0095http://refhub.elsevier.com/S0009-2797(14)00251-8/h0095http://refhub.elsevier.com/S0009-2797(14)00251-8/h0100http://refhub.elsevier.com/S0009-2797(14)00251-8/h0100http://refhub.elsevier.com/S0009-2797(14)00251-8/h0100http://refhub.elsevier.com/S0009-2797(14)00251-8/h0105http://refhub.elsevier.com/S0009-2797(14)00251-8/h0105http://refhub.elsevier.com/S0009-2797(14)00251-8/h0110http://refhub.elsevier.com/S0009-2797(14)00251-8/h0110http://refhub.elsevier.com/S0009-2797(14)00251-8/h0115http://refhub.elsevier.com/S0009-2797(14)00251-8/h0115http://refhub.elsevier.com/S0009-2797(14)00251-8/h0115http://refhub.elsevier.com/S0009-2797(14)00251-8/h0115http://refhub.elsevier.com/S0009-2797(14)00251-8/h0120http://refhub.elsevier.com/S0009-2797(14)00251-8/h0120http://refhub.elsevier.com/S0009-2797(14)00251-8/h0120http://refhub.elsevier.com/S0009-2797(14)00251-8/h0125http://refhub.elsevier.com/S0009-2797(14)00251-8/h0125http://refhub.elsevier.com/S0009-2797(14)00251-8/h0130http://refhub.elsevier.com/S0009-2797(14)00251-8/h0130http://refhub.elsevier.com/S0009-2797(14)00251-8/h0135http://refhub.elsevier.com/S0009-2797(14)00251-8/h0135http://refhub.elsevier.com/S0009-2797(14)00251-8/h0135http://refhub.elsevier.com/S0009-2797(14)00251-8/h0135http://refhub.elsevier.com/S0009-2797(14)00251-8/h0135http://refhub.elsevier.com/S0009-2797(14)00251-8/h0140http://refhub.elsevier.com/S0009-2797(14)00251-8/h0140http://refhub.elsevier.com/S0009-2797(14)00251-8/h0145http://refhub.elsevier.com/S0009-2797(14)00251-8/h0145http://refhub.elsevier.com/S0009-2797(14)00251-8/h0150http://refhub.elsevier.com/S0009-2797(14)00251-8/h0150http://refhub.elsevier.com/S0009-2797(14)00251-8/h0150http://refhub.elsevier.com/S0009-2797(14)00251-8/h0150http://refhub.elsevier.com/S0009-2797(14)00251-8/h0155http://refhub.elsevier.com/S0009-2797(14)00251-8/h0155http://refhub.elsevier.com/S0009-2797(14)00251-8/h0160http://refhub.elsevier.com/S0009-2797(14)00251-8/h0160http://refhub.elsevier.com/S0009-2797(14)00251-8/h0160http://refhub.elsevier.com/S0009-2797(14)00251-8/h0160http://refhub.elsevier.com/S0009-2797(14)00251-8/h0160http://refhub.elsevier.com/S0009-2797(14)00251-8/h0160http://refhub.elsevier.com/S0009-2797(14)00251-8/h0155http://refhub.elsevier.com/S0009-2797(14)00251-8/h0155http://refhub.elsevier.com/S0009-2797(14)00251-8/h0150http://refhub.elsevier.com/S0009-2797(14)00251-8/h0150http://refhub.elsevier.com/S0009-2797(14)00251-8/h0150http://refhub.elsevier.com/S0009-2797(14)00251-8/h0150http://refhub.elsevier.com/S0009-2797(14)00251-8/h0145http://refhub.elsevier.com/S0009-2797(14)00251-8/h0145http://refhub.elsevier.com/S0009-2797(14)00251-8/h0140http://refhub.elsevier.com/S0009-2797(14)00251-8/h0140http://refhub.elsevier.com/S0009-2797(14)00251-8/h0135http://refhub.elsevier.com/S0009-2797(14)00251-8/h0135http://refhub.elsevier.com/S0009-2797(14)00251-8/h0135http://refhub.elsevier.com/S0009-2797(14)00251-8/h0130http://refhub.elsevier.com/S0009-2797(14)00251-8/h0130http://refhub.elsevier.com/S0009-2797(14)00251-8/h0125http://refhub.elsevier.com/S0009-2797(14)00251-8/h0125http://refhub.elsevier.com/S0009-2797(14)00251-8/h0120http://refhub.elsevier.com/S0009-2797(14)00251-8/h0120http://refhub.elsevier.com/S0009-2797(14)00251-8/h0120http://refhub.elsevier.com/S0009-2797(14)00251-8/h0115http://refhub.elsevier.com/S0009-2797(14)00251-8/h0115http://refhub.elsevier.com/S0009-2797(14)00251-8/h0115http://refhub.elsevier.com/S0009-2797(14)00251-8/h0115http://refhub.elsevier.com/S0009-2797(14)00251-8/h0110http://refhub.elsevier.com/S0009-2797(14)00251-8/h0110http://refhub.elsevier.com/S0009-2797(14)00251-8/h0105http://refhub.elsevier.com/S0009-2797(14)00251-8/h0105http://refhub.elsevier.com/S0009-2797(14)00251-8/h0100http://refhub.elsevier.com/S0009-2797(14)00251-8/h0100http://refhub.elsevier.com/S0009-2797(14)00251-8/h0100http://refhub.elsevier.com/S0009-2797(14)00251-8/h0095http://refhub.elsevier.com/S0009-2797(14)00251-8/h0095http://refhub.elsevier.com/S0009-2797(14)00251-8/h0090http://refhub.elsevier.com/S0009-2797(14)00251-8/h0090http://refhub.elsevier.com/S0009-2797(14)00251-8/h0090http://refhub.elsevier.com/S0009-2797(14)00251-8/h0085http://refhub.elsevier.com/S0009-2797(14)00251-8/h0085http://refhub.elsevier.com/S0009-2797(14)00251-8/h0085http://refhub.elsevier.com/S0009-2797(14)00251-8/h0085http://refhub.elsevier.com/S0009-2797(14)00251-8/h0085http://refhub.elsevier.com/S0009-2797(14)00251-8/h0080http://refhub.elsevier.com/S0009-2797(14)00251-8/h0080http://refhub.elsevier.com/S0009-2797(14)00251-8/h0080http://refhub.elsevier.com/S0009-2797(14)00251-8/h0080http://refhub.elsevier.com/S0009-2797(14)00251-8/h0080http://refhub.elsevier.com/S0009-2797(14)00251-8/h0080http://refhub.elsevier.com/S0009-2797(14)00251-8/h0080http://refhub.elsevier.com/S0009-2797(14)00251-8/h0080http://refhub.elsevier.com/S0009-2797(14)00251-8/h0080http://refhub.elsevier.com/S0009-2797(14)00251-8/h0075http://refhub.elsevier.com/S0009-2797(14)00251-8/h0075http://refhub.elsevier.com/S0009-2797(14)00251-8/h0070http://refhub.elsevier.com/S0009-2797(14)00251-8/h0070http://refhub.elsevier.com/S0009-2797(14)00251-8/h0070http://refhub.elsevier.com/S0009-2797(14)00251-8/h0070http://refhub.elsevier.com/S0009-2797(14)00251-8/h0065http://refhub.elsevier.com/S0009-2797(14)00251-8/h0065http://refhub.elsevier.com/S0009-2797(14)00251-8/h0065http://refhub.elsevier.com/S0009-2797(14)00251-8/h0065http://refhub.elsevier.com/S0009-2797(14)00251-8/h0060http://refhub.elsevier.com/S0009-2797(14)00251-8/h0060http://refhub.elsevier.com/S0009-2797(14)00251-8/h0060http://-/?-http://refhub.elsevier.com/S0009-2797(14)00251-8/h0055http://refhub.elsevier.com/S0009-2797(14)00251-8/h0055http://refhub.elsevier.com/S0009-2797(14)00251-8/h0055http://-/?-http://refhub.elsevier.com/S0009-2797(14)00251-8/h0050http://refhub.elsevier.com/S0009-2797(14)00251-8/h0050http://refhub.elsevier.com/S0009-2797(14)00251-8/h0050http://refhub.elsevier.com/S0009-2797(14)00251-8/h0050http://refhub.elsevier.com/S0009-2797(14)00251-8/h0050http://-/?-http://refhub.elsevier.com/S0009-2797(14)00251-8/h0045http://refhub.elsevier.com/S0009-2797(14)00251-8/h0045http://refhub.elsevier.com/S0009-2797(14)00251-8/h0045http://refhub.elsevier.com/S0009-2797(14)00251-8/h0040http://refhub.elsevier.com/S0009-2797(14)00251-8/h0040http://refhub.elsevier.com/S0009-2797(14)00251-8/h0040http://refhub.elsevier.com/S0009-2797(14)00251-8/h0035http://refhub.elsevier.com/S0009-2797(14)00251-8/h0035http://refhub.elsevier.com/S0009-2797(14)00251-8/h0035http://refhub.elsevier.com/S0009-2797(14)00251-8/h0035http://refhub.elsevier.com/S0009-2797(14)00251-8/h0035http://refhub.elsevier.com/S0009-2797(14)00251-8/h0030http://refhub.elsevier.com/S0009-2797(14)00251-8/h0030http://refhub.elsevier.com/S0009-2797(14)00251-8/h0030http://refhub.elsevier.com/S0009-2797(14)00251-8/h0025http://refhub.elsevier.com/S0009-2797(14)00251-8/h0025http://refhub.elsevier.com/S0009-2797(14)00251-8/h0020http://refhub.elsevier.com/S0009-2797(14)00251-8/h0020http://refhub.elsevier.com/S0009-2797(14)00251-8/h0015http://refhub.elsevier.com/S0009-2797(14)00251-8/h0015http://refhub.elsevier.com/S0009-2797(14)00251-8/h0015http://refhub.elsevier.com/S0009-2797(14)00251-8/h0015http://refhub.elsevier.com/S0009-2797(14)00251-8/h0015http://refhub.elsevier.com/S0009-2797(14)00251-8/h0015http://refhub.elsevier.com/S0009-2797(14)00251-8/h0010http://refhub.elsevier.com/S0009-2797(14)00251-8/h0010http://refhub.elsevier.com/S0009-2797(14)00251-8/h0005http://refhub.elsevier.com/S0009-2797(14)00251-8/h0005http://-/?-http://-/?-http://dx.doi.org/10.1016/j.cbi.2014.09.003http://-/?-

astin b

Documents