Dioscin, a natural steroid saponin, shows remarkable protective

Toxicology Letters 214 (2012) 69–80
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Toxicology Letters
journal homepage: www.elsevier.com/locate/toxlet
Dioscin, a natural steroid saponin, shows remarkable protective effect against
acetaminophen-induced liver damage in vitro and in vivo
Xiaoming Zhao a , Xiaonan Cong a , Lingli Zheng b , Lina Xu a , Lianhong Yin a , Jinyong Peng a,∗
a
b
College of Pharmacy, Dalian Medical University, 9 Western Lvshun South Road, Dalian 116044, China
The First Affiliated Hospital of Dalian Medical University, Dalian 116011, China
h i g h l i g h t s
The protective effect of dioscin against APAP-induced liver damage was investigated.
Dioscin shows remarkable protective effect against the damage in vitro and in vivo.
The protective effect of dioscin was through adjusting mitochondrial function.
Dioscin should be developed as a new drug for the treatment of liver injury.
a r t i c l e
i n f o
Article history:
Received 15 June 2012
Received in revised form 31 July 2012
Accepted 6 August 2012
Available online 21 August 2012
Keywords:
Acetaminophen
Dioscin
Hepatotoxicity
Liver damage
Mitochondrial
a b s t r a c t
The aim of the study was to investigate the protective effect of dioscin against APAP-induced hepatotoxicity. In the in vitro tests, HepG2 cells were given APAP pretreatment with or without dioscin. In the
in vivo experiments, mice were orally administrated dioscin for five days and then given APAP. Some
biochemical and morphology parameters were assayed and the possible mechanism was investigated.
Dioscin improved AST release, mitochondrial dysfunction, apoptosis and necrosis of HepG2 cells induced
by APAP. Following administration of dioscin, APAP-induced hepatotoxicity in mice was significantly
attenuated. Furthermore, the liver cell apoptosis and necrosis, and hepatic mitochondrial edema were
also prevented. Fifteen differentially expressed proteins were found by using proteomics, and six of them,
Suox, Krt18, Rgn, Prdx1, MDH and PNP were validated. These proteins may be involved in the hepatoprotective effect of dioscin and might cooperate with the levels of Ca2+ in mitochondria, decreased expression
of ATP2A2, and decreased mitochondrial cardiolipin. In addition, dioscin inhibited APAP-induced activation and expression of CYP2E1, up-regulated the expression of Bcl-2 and Bid, and inhibited the expression
of Bax, Bak and p53. Dioscin showed a remarkable protective effect against APAP-induced hepatotoxicity by adjusting mitochondrial function. These results indicated that dioscin has the capability on the
treatment of liver injury.
© 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Abbreviations: APAP, acetaminophen; ALT, alanine aminotransferase; AST,
aspartate aminotransferase; GSH, glutathione; GSSG, glutathione disulfide;
MDA, malondi-aldehyde; NAC, N-acetylcysteine; MALDI-TOF/TOF-MS/MS, matrixassisted laser desorption/ionization time of flight mass spectrometry; 2DE, two-dimensional gel electrophoresis; SDS-PAGE, sodium dodecyl sulfate
polyacrylamide gel electrophoresis; MTT, 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5di-phenytetrazoliumromide; TEM, transmission electron microscope; TUNEL,
TdT-mediated dUTP nick-end labeling; DAPI, 4 ,6 -diamidino-2-phenylindole;
AO/EB, acridine orange/ethidium bromide; JC-1, 5,5 ,6,6 -tetra-chloro-1,1 ,3,3 tetraethyl-imidacarbocyanine iodide; DAB, 3,3 -diaminobenzidine; Suox, sulfite
oxidase; Krt18, cytoskeletal 18; Rgn, regucalcin; Prdx1, peroxiredoxin-1; MDH,
malate dehydrogenase; PNP, purine nucleoside phosphorylase.
∗ Corresponding author. Tel.: +86 411 8611 0411; fax: +86 411 8611 0411.
E-mail address: [email protected] (J. Peng).
0378-4274/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.toxlet.2012.08.005
Acetaminophen (APAP) is a safe and effective analgesic and
antipyretic drug at therapeutic doses. However, an overdose of
APAP can result in severe liver injury (Savides and Oehme, 1983).
The mechanism of APAP-induced liver injury has been studied for
several decades. Early results revealed the formation of a reactive metabolite (N-acetyl-p-benzoquinone imine, NAPQI), which is
responsible for liver injury through depletion of glutathione (GSH)
and binds to cellular proteins (Mitchell et al., 1973). Hepatotoxicity induced by APAP is considered to involve liver cytochrome
P450s (CYPs) including CYP2E1, CYP3A4 and CYP1A2 (Lucas et al.,
2005). According to reports, APAP-induced liver injury involves
many cell organelles, where mitochondrial damage is the most
important element (Ni et al., 2012), and mitochondrial dysfunction
has been identified as a pivotal mechanism (Labbe et al., 2008). The
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X. Zhao et al. / Toxicology Letters 214 (2012) 69–80
mitochondrion is the organelle which produces energy, and
plays a key role in controlling either cell survival or death
(Latchoumycandance et al., 2007). NAPQI, the toxic metabolite of
APAP, inhibits mitochondrial oxidative phosphorylation (Meyers
et al., 1988), depletes adenosine triphosphate (ATP), produces
selective mitochondrial oxidant stress (Jaeschke, 1990), decreases
mitochondrial membrane potential, and increases Ca2+ levels
(Holownia and Braszko, 2004). APAP can also cause alterations
in membrane permeability transition and release pro-apoptotic
factors into the cytosol. These are accompanied by caspase-3 activation, DNA fragmentation and apoptotic/necrotic cell death (Cover
et al., 2005; Kon et al., 2004; Ramachandran et al., 2011). A concept has emerged that mitochondrial dysfunction and damage is a
central event responsible for liver injury caused by APAP.
Faced with the threat of liver damage caused by APAP, scientists and pharmacists have attempted to find effective chemicals
or drugs to treat APAP-induced hepatotoxicity. In the 1970s,
N-acetylcystenine (NAC) was introduced to treat patients with
APAP-induced liver failure. NAC is still used in the clinic today
(Sandilands and Bateman, 2009), and is the only drug approved
for the treatment of APAP poisoning (Bajt et al., 2004). However,
NAC in the treatment of APAP-induced liver damage has considerable limitations, because the therapeutic window of this drug is
quite narrow. Thus, it is urgent and critically important to identify
and explore effective drugs to treat APAP-induced hepatotoxicity
(Yin et al., 2009). Natural products (NPs), one of the most important
resources in drug discovery, have been widely investigated worldwide and have the advantages of abundant resources (Saleem et al.,
2010). Due to the advantages of NPs, more and more researchers
have focused on these chemicals, and many natural chemicals
including silymarin (Wagoner et al., 2010), liquiritigenin (Kim et al.,
2006) and arjunolic acid (Manna et al., 2007) are considered to be
potential chemicals for the treatment of liver injury.
Dioscin (Fig. 1A) (Yin et al., 2010), which represents a typical steroid saponin, has been isolated from a number of oriental
vegetables and medicinal plants. Pharmacolo-gical studies have
shown that dioscin has anti-tumor (Wang et al., 2006), antihyperlipidemic (Li et al., 2010), and anti-fungal activities (Li et al.,
2003). Furthermore, this compound can be hydrolyzed to diosgenin, which has been widely used to synthesize many useful
steroid hormones and contraceptive drugs (Gomez et al., 2004). In
our previous study, we found that dioscin demonstrated a remarkable protective effect against CCl4 -induced acute liver damage in
mice, and the possible mechanism of action was also investigated
based on proteomic and bioinformatic techniques (Lu et al., 2011,
2012). However, the protective effect of dioscin against APAPinduced liver damage has not been reported in our best knowledge.
The aim of the present study was to investigate the protective effect of dioscin against APAP-induced liver injury in vitro
and in vivo. A significant hepatoprotective effect of the natural product against APAP-induced liver injury was demonstrated,
and two-dimensional gel electrophoresis (2-DE) coupled with
Matrix-assisted laser desorption/ionization time of flight mass
spectrometry (MALDI-TOF/TOF-MS/MS) was then used to investigate the mechanism of the action. Our results indicate that the
effect of dioscin against APAP-induced hepatotoxicity was through
the regulation of mitochondrial function, and this natural compound should be developed as a new drug for the treatment of liver
damage in the future.
2. Materials and methods
silybinin (57.3%), silydianin (2.5%) and silychristine (21.2%). APAP with the purity of
>98% was purchased from HEOWNS (HEOWNS, China).
2.2. Cell culture
HepG2 cells were obtained from the Institute of Biochemistry Cell Biology (IBCB,
CAS, China). The cells were cultured in minimum essential medium supplemented
with 10% fetal bovine serum (FBS) at 37 ◦ C in 5% CO2 . In all experiments, cells were
allowed to adhere and grow for 24 h in the culture medium prior to treatment.
2.3. Cell viability assay
Cells were seeded in a 96-well plate with 100 ␮L (1 × 105 cells/mL) per well, and
then allowed to adhere and grow for 24 h. Cells were pre-incubated with dioscin
(0.65, 1.3 and 2.6 ␮g/mL) for 6 h and then APAP (0, 5, 10 and 20 mM) was added for
24 h. Cell viability was then evaluated using the MTT assay (Solarbio Beijing, China).
The absorbance of samples was quantified at the wavelength of 570 nm using a
spectrophotometer (Thermo, USA).
2.4. Mitochondrial membrane potential assay
HepG2 cells was assessed using the 5,5 ,6,6 -tetrachloro-1,1 ,3,3 -tetraethylimidacar-bocyanine iodide (JC-1) kit according to the manufacturer’s instructions
(Beyotime Institute of Biotechnology, China). In normal cells, JC-1 was preferentially
localized in the mitochondria, where it formed aggregates with red fluorescence.
When the mitochondrial membrane potential declined, the dye diffused into the
cytosol in the monomeric form with green fluorescence. Thus, mitochondrial membrane potential was analyzed by the fluorescent images obtained using a laser
scanning confocal microscope (Leica, TCS SP5, Germany) and the fluorescence
intensity was determined by a fluorimeter (F-7000, Hitachi High-Technologies
Corporation, Japan) using an emission wavelength of 590 nm and an excitation
wavelength of 525 nm.
2.5. Acridine orange/ethidium bromide (AO/EB) assay
The cells were treated with APAP or dioscin as described above. The treated
HepG2 cells were then seeded on slides and stained according to the examination
kit (Kaiji Biological Technology, Nanjing, Jiangsu, China). Images of the cells were
obtained using a fluorescence microscope (OLYMPUS, Japan).
2.6. Comet assay
HepG2 cells were seeded in a 6-well cell culture plate at a density of
1 × 105 cells/mL. Then the cells were exposed to dioscin or APAP using the same
method as described above. The comet assay was performed under alkaline conditions according to the manufacturer’s instructions (CELL BIOLABS, INC., USA). Images
of the cells were obtained using a fluorescence microscope (OLYMPUS, Japan). At
least 150 randomly selected cells (50 cells from each of the three replicate slides)
were analyzed per sample with the Comet Assay Software Project (CASP) 1.2.2.
2.7. Animal experiments
Kunming male mice (18–22 g) were obtained from the Experimental Animal
Center of Dalian Medical University (Dalian, China, Quality certificate number: SCXK
(Liao) 2008-0002). Animals were housed 10 mice per cage and maintained in a
controlled environment at 25 ± 2 ◦ C under a 12 h dark/light cycle, and acclimatized for at least one week prior to the experiments. Dioscin suspended in 0.5%
carboxymethylcellulose sodium (CMC-Na) was administered intragastrically (i.g.)
at 25, 50, 100 mg/kg once daily for 5 consecutive days. Silymarin (200 mg/kg) was
administered (i.g.) as the positive control (Sharma et al., 2011). The animals in the
control and model groups were administered appropriate vehicles. Two hours after
the final treatment, the mice were injected with 300 mg/kg APAP intraperitoneally
(i.p.) (Yin et al., 2009). Twenty-two hours after APAP administration, the mice were
sacrificed and blood was collected. Serum samples were obtained by centrifugation
(3000 × g, 4 ◦ C) for 10 min and frozen at −80 ◦ C until assayed. After the animals were
killed, the livers were promptly removed and weighed. A portion of the liver was
fixed, and the remaining tissues were stored at −80 ◦ C. Animal experiments were
performed according to the guidelines of the Animal Care and Use Committee.
2.8. Biochemical assay
The activities of alanine aminotransferase (ALT), aspartate aminotransferase
(AST) and the contents of malondialdehyde (MDA), GSH, glutathione disulfide
(GSSG) were measured according to the kit instructions (Oxis International, Portland, OR, USA).
2.1. Materials
2.9. H&E assay
Dioscin with the purity of >98% was obtained from the National Institute for
the Control of Pharmaceutical and Biological Products (Beijing, China). Silymarin
was purchased from Sigma Chemical Co. (Sigma Co., Milan, Italy), and contained
After the mice were sacrificed, the liver tissue was removed and a portion of
the tissue was instantly fixed in 10% formalin, embedded in paraffin, cut into 5 ␮m
X. Zhao et al. / Toxicology Letters 214 (2012) 69–80
71
Fig. 1. (A) The chemical structure of dioscin. (B) HepG2 cells were treated with different concentrations of dioscin and APAP. Cell viability was determined by MTT assay.
JC-1, AO/EB and DAPI staining were analyzed in control, APAP (20 mM) and APAP + dioscin (20 mM and 1.3 ␮g/mL) treated groups. (C) Mitochondrial membrane potential
of cells detected by JC-1 assay. The fluorescence images of JC-1 for cells (400×, final magnification). (D) Representative images of AO/EB stained HepG2 cells (200×, final
magnification). After APAP treatment, apoptotic cells (orange fluorescence) exhibited chromatin condensation and apoptotic bodies (arrows). (E) Representative images of
DAPI stained HepG2 cells (200×, final magnification). Data were presented as mean ± S.E.M. **p < 0.01 compared with APAP group.
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X. Zhao et al. / Toxicology Letters 214 (2012) 69–80
pieces and mounted on slides. The samples were stained with hematoxylin and eosin
(H&E) for histopathological examination.
2.10. DAPI assay
Mice and HepG2 cells were treated with dioscin/APAP or APAP as described
above. Nuclear staining with 4 ,6 -diamidino-2-phenylindole (DAPI) was performed
to evaluate apoptosis. Liver paraffin sections and fixed cells were permeabilized
with 0.5% Triton X-100 for 5 min followed by incubation with DAPI (1 ␮g/mL) for
8 min (Sigma, USA), then washed and examined by fluorescence microscopy (CKX41,
OLYMPUS, Japan).
2.11. TUNEL assay
Mice and HepG2 cells were treated with dioscin/APAP or APAP as described
above. Liver paraffin sections were prepared and the cells were fixed on slides. Then
they were used for the TdT-mediated dUTP Nick-End Labeling (TUNEL) assay, which
was performed using a commercial kit according to the manufacturer’s instructions (In Situ Cell Death Detection Kit, Fluorescein, Roche). Briefly, the fluorescein
(green) labeled dUTP solution was added on the surface of slides and incubated at
37 ◦ C for 1 h. Then the sections were washed and photographed using fluorescence
microscopy (CKX41, OLYMPUS, Japan). 50 ␮L converter-POD was added on the samples for the reaction at 37 ◦ C for 30 min and washed. Then the sections were stained
with 3,3 -diaminoben-zidine tetrahydrochloride (DAB) fluid. Images were obtained
using inverted digital imaging light microscopy (Nikon Eclipse TE2000-U, NIKON,
Japan). After the staining, TUNEL positive cells showed green fluorescence or brown
staining.
2.12. Transmission electron microscope (TEM) assay
Mice and HepG2 cells were treated with dioscin/APAP or APAP as described
above. The pre-treatment of samples was carried out as previously described
(Beloosh et al., 2010). Regions of interest were excised with a glass scribe and
mounted for ultramicrotomy. The sections were then stained and observed using a
transmission electron microscope (JEM-2000EX, JEDL, Japan).
2.13. Measurement of mitochondrial damage
The method for separating mitochondria from the liver tissue was conducted
according to Tissue Mitochondria Isolation Kit (Beyotime Institute of Biotechnology, China). Briefly, 80 mg fresh liver tissue was made into 10% homogenate with
separating reagent and centrifuged at 600 × g, 4 ◦ C to produce the supernatant.
Then, the purified liver mitochondria were collected from the sediment after the
centrifugation of supernatant at 11000 × g, 4 ◦ C.
The amount of cardiolipin in the purified liver mitochondria was measured using
nonyl-acridine orange fluorescence according to the manufacturer’s instructions
(GENMED Institute of Medical Technology, Shanghai, China). The free calcium in
purified liver mitochondria was measured using FLUO-3AM fluorescence according
to the manufacturer’s instructions (Beyotime Institute of Biotechnology, China). The
fluorescence was measured using a fluorimeter (HITACHI, Japan).
2.14. Two-dimensional gel electrophoresis (2-DE)
Part of the livers from three mice in each group was washed three times, and
total protein was isolated using the tissue protein extraction kit according to the
manufacturer’s instructions (Bio-Rad, USA). Protein content in the supernatants was
determined by the Bradford assay. Samples were aliquoted and stored at −80 ◦ C until
proteomic analysis. Two-dimensional gel electrophoresis (GE Healthcare IPGphor
system) was used to separate the proteins. Total protein (100 ␮g) was determined in
first dimension isoelectric focusing (IEF) with non-linear gradient IPG-strips (13 cm),
pH 3–10 and a programmed voltage gradient. IPG-strips were rehydrated with the
sample at 30 V for 12 h, and then IEF was conducted at 500 V for 1 h, 1000 V for 1 h,
and then 8000 V for 10 h to reach a total of approximately 60 kVh. After this run was
complete, the strips were equilibrated by gentle shaking in two steps for 10 min each
in equilibration buffer I (1% dithiothreitol, 50 mM pH 6.8 Tris–hydrochloride, 6 M
Urea, 30% Glycerol, 2% SDS bromophenol blue) and equilibration buffer II (dithiothreitol was replaced with 2.5% iodoacetamide). The IPT strips were then placed
in 12.5% acrylamide gels in the second dimension at 30 mA for 3 h until the bromophenol blue front reached the bottom of the gel. The separated proteins were
visualized by silver diamine-staining. For preparative 2-DE, 800 ␮g of total proteins
were separated as described above. Proteins were detected by deep purple staining
(GE Healthcare) according to the manufacturer’s instructions. The silver-stained 2-D
gels were scanned by a GS-710 imaging densitometer. The digitized images were
analyzed by Imagine Master software (GE Healthcare). All processes were repeated
three times.
2.15. Protein identification
Significant changes in protein expression were considered when the differences were more than or equal to 2-fold as calculated by the staining intensity
of the stained plot. Interested spots with significant differences in the preparative gels were manually excised. The peptides in the gel samples were extracted
by trypsin digestion (Promega, Madison, WI, USA). The purified peptides were
spotted on a MALDI plate and covered with 0.7 mL of 2 mg/mL 3,5-dimethoxy-4hydroxycinnamic acid matrix (Sigma, USA) with 10 mM NH4 H2 PO4 in 60% ACN. All
samples were then analyzed by MALDI-TOF/TOF MS/MS with a 4800 Proteomics
Analyzer (Applied Biosystems, Foster City, CA, USA). Monoisotopic peak masses were
acquired in a mass range of 800–4,000 Da, with a signal-to-noise ratio (S/N) of 1/200.
Five of the most intense ion signals, excluding common trypsin autolysis peaks and
matrix ion signals, were selected as precursors for MS/MS acquisition. The peptide
mass fingerprint (PMF) combined MS/MS data were submitted to MASCOT version
2.1 (Matrix Science) for identification according to the Swiss Prot 2009.11 57.11 Rattus database. The criteria for a successfully identified protein were as follows: ion
score confidence interval (C.I.%) for PMF and MS/MS data ≥95%.
2.16. Activity and expression of CYP2E1
CYP2E1 activity was assessed in liver microsomes by measuring p-nitrophenol
hydroxylation as previous description (Kim et al., 1988). Microsomes were obtained
from livers after APAP treatment using the reported methods (Schenkman and
Jansson, 1999). Values were expressed as nmol/min/mg protein. The expression of
CYP2E1 was analyzed by western blotting method as follows.
2.17. Western blotting
For the Western blot assay, total protein was isolated using the tissue protein
extraction kit based on the manufacturer’s instructions (Bio-Rad, USA). An aliquot
(containing 50 ␮g protein) of the supernatant was loaded onto the SDS gel (10–12%),
separated electrophoretically, and transferred to a PVDF membrane (Millipore, USA).
After the PVDF membrane was incubated with 10 mM TBS plus 1.0% Tween 20 and 5%
dried skim milk (Boster Biological Technology, China) to block nonspecific protein
binding, the membrane was incubated overnight at 4 ◦ C with primary antibodies
including cytoskeletal 18 (Krt18, 1:500), regucalcin (Rgn, 1:1000), peroxiredoxin-1
(Prdx1, 1:200), sulfite oxidase (Soux, 1:500), Ca2+ -ATPase (ATP2A2, 1:500), CYP2E1
(1:1000) (Protein Tech Group, Wuhan, China), procaspase-3 (1:500), p53 (1:500), Bid
(1:200), Bcl-2 (1:200) (Santa, Japan), and glyceraldehyde phosphate dehydrogenase
(GAPDH, 1:2000) (Sigma, MO, USA). Blots were then incubated with horseradish
peroxidase-conjugated antibodies for 2 h at room temperature at a 1:2000 dilution (Beyotime Institute of Biotechnology, China). Detection was performed using
an enhanced chemiluminescence (ECL) method and photographed using the BioSpectrum Gel Imaging System (UVP, USA). To eliminate the variations due to protein
quantity and quality, the data were adjusted to GAPDH expression (IOD of objective
protein versus IOD of GAPDH protein).
2.18. Immunohistochemistry
Immunohistochemistry (IHC) was performed on 10% formalin-fixed and paraffin
-embedded tissues. Deparaffinized sections were incubated in 3% hydrogen peroxide (H2 O2 ) for 30 min and normal goat serum to block nonspecific protein binding
for 30 min. The sections were then incubated at 4 ◦ C overnight with rabbit anti-Bax
and anti-Bak antibodies at a 1:100 dilution (Protein Tech Group, Wuhan, China),
followed by incubation in PV6001 (horseradish peroxidase-conjugated goat antirabbit IgG) Two-Step IHC Detection Reagent for 30 min at 37 ◦ C. DAB, used as the
chromogen, was counterstained with hematoxylin. Images were obtained using
inverted digital imaging light microscopy (Nikon Eclipse TE2000-U, NIKON, Japan).
The brown-stained areas were considered positive.
2.19. ELISA
The concentrations of malate dehydrogenase (Mdh1) and purine nucleoside
phosphorylase (Pnp) in the serum of mice were measured according to the ELISA kit
instructions (Bluegene, China).
2.20. Statistical analysis
All the data were analyzed using statistical software SPSS 11.5. Data are
expressed as mean ± S.E.M. Multi-group comparisons were performed using the
one-way ANOVA test, while couple comparisons were performed using the t-test.
Differences were considered significant when p < 0.05.
3. Results
3.1. Protective effect of dioscin against APAP-induced
hepatotoxicity in vitro
To explore the cytoprotective effect of dioscin in vitro, we used
APAP-treated HepG2 cells as the liver injury model (McGill et al.,
2011). HepG2 cells were pre-treated with different concentrations
X. Zhao et al. / Toxicology Letters 214 (2012) 69–80
(0.65, 1.3 and 2.6 ␮g/mL) of dioscin and then treated with APAP. Different concentrations of APAP (5, 10 and 20 mM) were tested and
the results showed that 10 and 20 mM APAP could cause significant differences between APAP and dioscin treated groups (p < 0.01)
(Fig. 1B). While, the MTT assay showed that cell viability was
markedly increased from 54.1 ± 3.0% to 81.3 ± 1.2% when the cells
were pre-treated with dioscin (1.3 ␮g/mL) compared with the APAP
(20 mM) treated group. The cytoprotective effect of dioscin was
more markedly after the treatment of 20 mM APAP than 10 mM.
Therefore, 20 mM APAP was selected for subsequent experiments.
The MTT assay showed that cell viability was markedly
increased when cells were pre-treated with dioscin (1.3 ␮g/mL)
compared with the model group (p < 0.01). Dioscin at the higher
concentration of 2.6 ␮g/mL showed no cytotoxicity. But its effect
was equal to dioscin at 1.3 ␮g/mL (p < 0.01). Thus, dioscin at the concentration of 1.3 ␮g/mL was selected for subsequent investigations.
In addition, AST level in the dioscin-pretreated/APAP group was
significantly changed (p < 0.05) (Supplementary Fig. 1A) compared
with the group treated with APAP alone. However, GSH concentration was not different between the dioscin-pretreated and model
groups (Supplementary Fig. 1B), which suggested that dioscin does
not directly increase intracellular GSH level.
Representive fluorescent images of mitochondrial membrane
potential detected by JC-1 were presented in Fig. 1C. Compared
with the APAP-treated group, there was a clear increase in red fluorescence in the control and dioscin-pretreated groups at the dose
of 1.3 ␮g/mL. The fluorescence of JC-1 aggregate was significantly
increased from 64.4 ± 3.0 in the APAP group to 75.7 ± 3.5 (p < 0.05)
in the group treated with 1.3 ␮g/mL of dioscin (Supplementary Fig.
1C). These results indicated that dioscin prevented the decrease of
mitochondrial membrane potential induced by APAP.
AO/EB staining was applied to investigate the effect of dioscin
on APAP-induced cell apoptosis or necrosis. As shown in Fig. 1D,
the normal cells in control showed green fluorescence. While the
orange fluorescence in HepG2 cells exposed to APAP (20 mM)
indicated the cell apoptosis with chromatin condensation and
apoptotic bodies, which were significantly improved by pretreatment with dioscin. Further study by DAPI staining as shown in
Fig. 1E, the cell nucleus chromatin was condensed and nuclear
apoptotic bodies were formed in the APAP-treated group. However,
in the dioscin pre-treated group, the cell nucleus was complete and
the chromatin was uniform.
In Fig. 2A, the result of comet assay was used to evaluate the
damage of cell nucleus. During electrophoresis, the normal nucleus
exhibited round fluorescence image in control cells, while DNA
fragment migration formed smears due to cell apoptosis and DNA
breakage induced by APAP. The results in Fig. 2C showed that the
Comet Length in the APAP-treated group (227 ± 8.1 ␮m) was much
longer than that in the dioscin-pretreated group (158 ± 5.2 ␮m)
(p < 0.01). In addition, apoptotic hepatocytes detected by TUNEL
assay indicated that dioscin significantly decreased the number
of TUNEL positive cells (Fig. 2B, D and E). Thus, dioscin strongly
prevented APAP-induced cell apoptosis.
Morphological observation of HepG2 cells by TEM indicated
that mitochondria were seriously damaged by APAP (Fig. 2F). The
mitochondrial double membrane was unclarity, distorted cristae
were depleted in number and the matrix had a reduced number of electron-dense granules. However, dioscin could remarkably
decrease these injury and protected mitochondria.
3.2. Protective effect of dioscin against APAP-induced
hepatotoxicity in vivo
To investigate the protective effect of dioscin against APAPinduced hepatotoxicity in vivo, male Kunming mice were
administered dioscin i.v. at the doses of 25, 50 and 100 mg/kg before
73
i.p. injection of 300 mg/kg APAP. We also examined the effect of
dioscin treatment alone at the dose of 100 mg/kg in mice. In the
APAP-treated group, serious liver injury occurred and was characterized by high serum levels of ALT and AST, which was prevented
by dioscin as demonstrated by a significant decrease in serum ALT
levels from 2507 ± 487 U/L (APAP group) to 213 ± 80 U/L (dioscin
100 mg/kg pre-treated group), and AST levels from 702 ± 186 U/L
(model group) to 103 ± 37 U/L (dioscin 100 mg/kg pre-treated
group), which were equivalent or even better than the effect produced by silymarin at the dose of 200 mg/kg. The results also
showed that the content of liver homogenate GSH was increased
and GSSG was decreased in the group pre-treated with dioscin at
the doses of 50 and 100 mg/kg (Table 1). Furthermore, the data
also showed that pretreatment with dioscin at the doses of 50
and 100 mg/kg decreased liver homogenate MDA level. Histological (H&E) evaluation further revealed that APAP-induced massive
bridging necrosis within the centralobular region of the liver was
significantly ameliorated by dioscin in a dose-dependent manner
(Fig. 3A). The DAPI assay showed that hepatocyte nucleus chromatin was condensed and a lot of particulate matter had formed in
the APAP-treated group (Fig. 3B). However, the hepatocyte nucleus
in the dioscin-pretreated group had a complete nuclear shape with
uniform chromatin.
The TUNEL assay was conducted to identify apoptotic and necrosis cells among the injured hepatocytes. In Fig. 3C, D and E, more
TUNEL-positive cells, with green fluorescence and brown staining,
were observed in the APAP-treated group than in the dioscinpretreated group (p < 0.01), which indicated that dioscin could
protect liver from hepatocyte apoptosis induced by APAP.
3.3. Effects of dioscin on mitochondrial dysfunction
Mitochondrial dysfunction was associated with structural
abnormalities. Mitochondrial swelling and loss of structural
integrity were induced by APAP. To determine the protective effect
of dioscin against APAP-induced mitochondrial dysfunction, we
measured mitochondrial morphology and function in mouse liver.
TEM observations of the nucleus and mitochondria in liver cells
were shown in Fig. 4A, which demonstrated that intact mitochondria were clearly visible at the double membrane, and intact cristae
structure and a mitochondrial matrix consisting of electron-dense
granules were found in control hepatocytes. While the mitochondrial double membrane was absent and leaky, distorted cristae
were depleted in number and the matrix had a reduced number
of electron-dense granules in the APAP treated group. However,
these findings were significantly ameliorated by the pretreatment
of dioscin. These results indicate that dioscin protected mitochondrial against APAP-induced injury.
In addition, the cardiolipin content of mitochondrial membrane was determined to evaluate the oxidative damage of the
mitochondria membrane and the fluorescence was measured by
a fluorimeter using an emission wavelength of 535 nm and excitation wavelength of 485 nm. As shown in Fig. 4B, cardiolipin
fluorescence was higher in the dioscin-pretreated group than
in the APAP-treated group. Ca2+ -ATPase plays a critical role in
maintaining the balance between cytosolic and mitochondrial
Ca2+ concentration (Moore et al., 1985). In the present study,
the level of mitochondrial Ca2+ was detected using FLUO-3 AM.
The fluorescence of Ca2+ in the model group was higher than
that in the control group, however, the high Ca2+ level in the
APAP-treated group was significantly decreased from 22.2 ± 0.7
to 14.5 ± 0.9 (p < 0.05) by dioscin in a dose-dependent manner (Fig. 4C). In addition, the expression of ATP2A2 in the
APAP-treated group was lower than that in the other groups,
and increased by 4.93-fold (p < 0.01) in the 100 mg/kg dioscinpretreated group (Fig. 4D). APAP decreased the expression of
74
X. Zhao et al. / Toxicology Letters 214 (2012) 69–80
Fig. 2. (A) Comet assay of the protective action of dioscin (1.3 ␮g/mL) against DNA damage in HepG2 cells induced by APAP (20 mM) (200×, final magnification). (B) TUNEL
positive cells were shown with green fluorescence and brown staining (100×, final magnification) after the treatment of dioscin (1.3 ␮g/mL) and APAP (20 mM). (C) Statistic
analysis of Comet length. (D) Statistic analysis of TUNEL positive cells after fluorescein staining. (E) Statistic analysis of TUNEL positive cells after DAB staining. (F) TEM
assay of the protective effect of dioscin (1.3 ␮g/mL) against structural damage of mitochondria induced by APAP (20 mM). Data were presented as mean ± S.E.M. **p < 0.01
compared with APAP group.
intracellular ATP2A2 and elevated the level of Ca2+ in mitochondria. These results suggested that dioscin protected against
APAP-induced liver injury by amelioration of mitochondrial function.
3.4. Validation of differentially expressed proteins
For a comprehensive evaluation of the effects of dioscin against
APAP-induced acute liver injury, liver proteins from the control,
APAP and dioscin-pretreated groups were submitted to 2-DE, and
the electrophoresis patterns are presented in Fig. 5A. Sixteen
differentially expressed proteins obtained by pairwise comparison (APAP-treated versus control groups, APAP-treated versus
DIO + APAP groups) were found and 15 were identified (Table 2).
We then selected six proteins including Soux, Krt18, Rgn, Prdx1,
Pnp and Mdh1 as the marker proteins for the subsequent study
based on Western blotting and ELISA. Compared with the model
group, the protein expression of Soux and Prdx1 (Fig. 5B and C)
in the dioscin-pretreated group increased by 1.65- (p < 0.05), 20.2fold(p < 0.01). Mdh1 and Pnp were also increased from 11.3 ± 0.5
X. Zhao et al. / Toxicology Letters 214 (2012) 69–80
75
Fig. 3. Effects of dioscin (100 mg/kg) on APAP (300 mg/kg)-induced liver injury in mice. (A) Hematoxylin and eosin (H&E) staining for histopathological examination (100×,
final magnification). (B) DAPI assay of the protective effect of dioscin against DNA damage in liver cells induced by APAP (200×, final magnification). Compared with normal
nuclei, APAP induced condensed nucleus chromatin, which was significantly ameliorated by dioscin (arrow indication). (C) The protective effect of dioscin against APAPinduced apoptosis in liver cells as determined by TUNEL assay. The cellular apoptosis indicated by green fluorescence and brown staining (100×, final magnification). (D)
Statistic analysis of TUNEL positive cells after fluorescein staining. (E) Statistic analysis of TUNEL positive cells after DAB staining. Data were presented as mean ± S.E.M.
**p < 0.01 compared with APAP group.
to 22.7 ± 0.43 ng/mL (p < 0.01) (Fig. 5D) and from 23.6 ± 0.91 to
37.0 ± 0.64 ng/mL (p < 0.01), respectively (Fig. 5E). The expression
of Rgn increased by 1.46-fold (p < 0.05) in the dioscin-pretreated
group (Fig. 5F), while the expression of Krt18 (Fig. 5G) decreased
by 1.53-fold (p < 0.05).
3.5. Dioscin altered mitochondrial pathway related proteins
APAP-induced hepatotoxicity is initiated by the formation of
NAPQI, which binds to cellular proteins and causes mitochondrial damage and oxidative stress culminating in hepatocyte death.
76
X. Zhao et al. / Toxicology Letters 214 (2012) 69–80
Fig. 4. (A) TEM assay of the protective effect of dioscin against structural damage of mitochondria induced by APAP (300 mg/kg). (B) The cardiolipin content of purified
mitochondria from the liver tissue in mice. (C) The Ca2+ content of purified mitochondria from the liver tissue in mice. (D) The APT2A2 expression detected by Western blot.
Data are presented as mean ± S.E.M. *p < 0.05 and **p < 0.01 compared with APAP group.
Fig. 5. (A) Representative 2-DE gel images of differentially expressed proteins in the liver of mice. Sixteen proteins (15–150 kDa) were detected under pH 3–10. Protein
expressions including Suox (B) and Prdx1 (C) were detected by Western Blot. Protein contents of Mdh1 (D) and Pnp (E) were determined by ELISA. Protein expressions of
Rgn (F) and Krt18 (G) were detected by Western Blot. Data are presented as mean ± S.E.M. *p < 0.05 and **p < 0.01 compared with APAP group.
X. Zhao et al. / Toxicology Letters 214 (2012) 69–80
77
Fig. 6. The activity (A) and expression (B) of CYP2E1 in mice liver. (C) The expression of p53 in mice liver. (D) The expression of Bax and Bak in mice liver determined
by immunohistochemistry (100×, final magnification) and their statistic analysis. The expression of Bcl-2 (E) and Bid (F) in mice liver. Data are presented as mean ± SEM.
*p < 0.05 and **p < 0.01 compared with model group.
CYP2E1 plays an important role in APAP metabolism (Zaher et al.,
1998). Thus, we investigated the activity and expression of CYP2E1
in mouse liver. The results showed that the activity and expression of CYP2E1 in the APAP-treated group was much higher
than that in the dioscin-pretreated group (Fig. 6A and B), which
suggested that the dioscin protected liver against APAP-induced
injury by decreasing the activity and down-regulating the expression of CYP2E1. Proteins related to the mitochondrial pathway
were also investigated to explore the possible action mechanism of
dioscin. Compared with the model group, the expression of p53 was
78
X. Zhao et al. / Toxicology Letters 214 (2012) 69–80
Table 1
Effects of dioscin on acetaminophen-induced liver injury.
ALT (U/L)
Control
Dioscin 100 mg/kg
APAP
Dioscin 100 mg/kg + APAP
Dioscin 50 mg/kg + APAP
Dioscin 25 mg/kg + APAP
Silymarin 200 mg/kg + APAP
23
25
2507
213
233
1640
570
±
±
±
±
±
±
±
3**
3**
487
80**
87**
215*
56**
AST (U/L)
40
45
702
103
109
633
336
±
±
±
±
±
±
±
2**
5**
186
37**
33**
161*
43*
GSH (nmol/L)
41
40
25
38
33
27
30
±
±
±
±
±
±
±
1.7**
1.8**
0.8
2.1**
1.3*
1.5
1.9*
GSSG (nmol/L)
0.5
0.5
1.9
0.8
0.9
1.7
1.2
±
±
±
±
±
±
±
0.08**
0.1**
0.09
0.2**
0.11*
0.13
0.1*
Mice were given APAP (300 mg/kg, i.p.) pretreatment with or without dioscin and silymarin, and sacrificed 22 h after the treatment. Data are presented as mean ± S.E.M.
(n = 10).
*
p < 0.05.
**
p < 0.01 compared with APAP group.
decreased by 1.53-fold (p < 0.05) in the dioscin-pretreated group at
the dose of 50 mg/kg (Fig. 6C). p53 can regulate proteins of the Bcl2 family to participate in cell apoptosis (Hassan et al., 2003). As
expected, dioscin significantly down-regulated the expression of
Bak and Bax (Fig. 6D), and up-regulated the expression of Bcl-2 and
Bid by 2.02- (p < 0.01) and 1.64-fold (p < 0.01) in a dose-dependent
manner in the high dose group compared with the model group
(Fig. 6E and F). These results indicated that the protective effect of
dioscin against APAP-induced liver injury was conducted by regulating mitochondrial pathway.
4. Discussion
An overdose of APAP causes liver injury in experimental animals
and humans. It is metabolized by the cytochrome P450-dependent
oxidative enzyme pathway to form a reactive intermediate metabolite, NAPQI, which conjugates with GSH and is further metabolized
(Dahlin et al., 1984). Subsequently, cells die over the next few hours
due to oxidative stress and opening of the mitochondrial permeability transition pore (Yasuhiro et al., 2005). These events lead to
a dramatic decline in mitochondrial bioenergetics and, ultimately,
cell death (Dahlin et al., 1984). CYP2E1 is the major enzyme of the
CYP450 isoenzyme in liver and metabolizes relatively few drugs.
The activity of CYP2E1 was increased following APAP treatment
(Lucas et al., 2005). In our study, the activity and expression of
CYP2E1 was inhibited by dioscin, which suggests that the protective
effect of dioscin is possibly caused by altered APAP bioactivation.
The present study showed that pretreatment with dioscin was
effective against APAP-induced hepatotoxicity. In the in vitro tests,
dioscin prevented decreased viability of HepG2 cells exposed to
APAP (Fig. 1). However, there was no significant change in cell GSH
level. To evaluate the role of dioscin in cell death induced by APAP,
several assays including the AO/EB, DAPI, TUNEL and SCGE assays
were employed and demonstrated that dioscin effectively prevented APAP-induced cell apoptosis and necrosis. These data show
that dioscin was able to protect HepG2 cells from APAP-induced
toxicity.
Because data on in vitro APAP toxicity cannot always be extrapolated to an in vivo situation, dioscin was administered orally to
mice. The in vivo data showed that dioscin significantly changed
some of the parameters of APAP toxicity including ALT, AST, MDA,
and GSH as shown in Table 1. In addition, H&E, TUNEL and DAPI
assays showed that dioscin strongly prevented APAP-induced
cell apoptosis or necrosis in the liver. The development of mitochondrial dysfunction has been observed following APAP toxicity,
and includes a decrease in membrane potential and Ca2+ -ATP
levels, and an increase in reactive oxygen species (ROS) and a
disequilibrium of Ca2+ (Moore et al., 1985). Our data demonstrate
that dioscin can protect against mitochondrial dysfunction and
damage caused by APAP. Thus, we think that the mechanism of
action of dioscin in protecting against APAP-induced liver injury is
through the regulation of mitochondrial function.
To investigate this mechanism, 2-DE coupled with MALDITOF/TOF-MS/MS was performed to find the protein markers and
fifteen proteins including Soux, Krt18, Rgn, Prdx1, Mdh1 and Pnp
were identified (Table 2).
Suox, a molybdenum-containing enzyme located in the intermembranous area of mitochondria, can oxidize sulfite to sulfate
(Johnson et al., 1980; Cohen et al., 1971). Sulfite is detoxified in the
liver by Suox and oxidized to sulfate (Oshino, 1975). When there
is a lack of Suox or the activity of the enzyme is decreased, most
of the sulfite cannot be catalyzed into sulfate. Sulfite is a systemic
toxic factor and can cause many diseases (Heimberg et al., 1953;
Claiborne et al., 2001). In the present study, we found that APAP
down-regulated the expression of Suox. Compared with the model
group, dioscin up-regulated the expression of Suox, which may be
related to the protection of mitochondria.
Prdx1 is highly expressed in bone marrow, brain, heart, liver
and ovary (Chae et al., 1999; Manevich et al., 2002). Overexpression of Prdx1 protects cells against antioxidant-induced plasma
membrane damage and apoptosis (Pak et al., 2002). Prdx1 was
up-regulated by dioscin following treatment with APAP in the
present study. Among the stress/oxidative stress responsive elements, mitochondrial cardiolipin molecules are likely targets of
oxygen free radical attack (Paradies et al., 2011). Peroxidation of
membrane lipid components has been hypothesized to be a major
mechanism of oxygen-free radical toxicity (Slater, 1984). Our data
showed that dioscin can elevate the content of mitochondrial cardiolipin. APAP induces oxidative stress in liver cells, leading to
significant changes in the redox balance. The increase in Prdx1
further suggests that dioscin has a protective effect against APAPinduced mitochondrial oxidative damage.
Traditionally, cytokeratins were considered only as skeletal proteins providing mechanical stability. However, recent evidence has
shown that they also have non-skeletal functions (Omary et al.,
2004). It was reported that Krt 18 induction occurs in response
to the oxidative damage generated by inorganic arsenic (iAs) and
should be considered as an early indicator of iAs toxicity in the liver
(Gonsebatt et al., 2007). Oxidative damage often occurs in mitochondria. Mitochondrial functional integrity is much less affected
by ROS administration in K8/K18-lacking hepatic cells (Mathew
et al., 2008). Altered Krt 18 expression could induce hepatic susceptibility to further toxic injury. Our data provide evidence that
dioscin suppresses APAP-induced Krt 18 overexpression, which
suggests that dioscin may adjust the expression of Ktr 18 to protect
against APAP-induced mitochondrial damage.
Rgn is mainly expressed in the liver (Shimokawa and
Yamaguchi, 1992). The expression of hepatic Rgn is mediated
through Ca2+ signaling factors (Murata and Yamaguchi, 1999) and
Rgn has a role in the regulation of Ca2+ -ATPase activity in mitochondria (Tsurusaki and Yamaguchi, 2000). Calcium deregulation has a
X. Zhao et al. / Toxicology Letters 214 (2012) 69–80
79
Table 2
The differentially expressed proteins in mice liver identified by proteomic method.a
Protein name
Gene name
Accession no.
MW
PI
Protein score
Protein score
C.I.%
Sulfite oxidasec
Cytoskeletal 18b
Coronin-1Ab
Gm9819 Putative uncharacterized proteinc
Regucalcinc
Inorganic pyrophosphatasec
Heme-binding protein 1c
Abhydrolase domain-containing protein 14Bc
Peroxiredoxin-1c
Purine nucleoside phosphorylasec
Malate dehydrogenasec
Fructose-1,6-bisphosphatase 1c
Glycerol-3-phosphate dehydrogenase [NAD+]c
Sorb 40 kDa proteinc
Phenazine biosynthesis-like domain-containing protein 2c
SUOX
Krt18
Corola
Gm9819
Rgn
Ppa1
Hebp1
Abhd14b
Prdx1
Pnp
Mdh1
Fbp1
Gpd1
Sorb 40 kDa protein
3110049J23Rik
IPI00153144
IPI00311493
IPI00323600
IPI00622968
IPI00133456
IPI00110684
IPI00135085
IPI00111876
IPI00121788
IPI00315452
IPI00336324
IPI00228630
IPI00230185
IPI00875416
IPI00110528
61231
47509.2
51641.2
32931.5
33898.7
33102.3
21153.4
22550.6
22390.4
32541.2
36659.1
37287.9
38175.7
40635.8
32191.4
6.07
5.22
6.05
4.86
5.15
5.37
5.18
5.82
8.26
5.78
6.16
6.15
6.75
6.6
5.19
640
990
96
697
835
710
588
606
846
979
691
654
666
652
706
100
100
99.999
100
100
100
100
100
100
100
100
100
100
100
100
a
b
c
The expressed proteins have significant differences between APAP-treated group versus control group.
Increased of the differentially expressed proteins between APAP-treated group versus control group.
Decreased of the differentially expressed proteins between APAP-treated group versus control group.
profound effect on cell survival via numerous signal-transduction
pathways utilizing Ca2+ as a second messenger (Yamaguchi et al.,
2008). An overdose of APAP induces cellular responses driven by
disruption of Ca2+ homeostasis and is strongly correlated to DNA
damage in the liver (Holownia and Braszko, 2004). This evidence is
consistent with our results that APAP can increase Ca2+ level and
decrease the expression of Ca2+ -ATPase. We also found that dioscin
can reverse these changes, which means that dioscin mediates Ca2+
balance through regulation of Rgn.
According to the proteomics findings, cytoplasmic Mdh1 levels were lowest in the APAP group than in the other groups.
Mdh1 catalyzes the conversion of oxaloacetate and malate utilizing the NAD+ /NADH coenzyme system, and participates in the
malate/aspartate shuttle (Minarik et al., 2002). Enzymes involved
in glucose metabolism change when liver cells are injured, which
results in poor function of the aerobic oxidation of glucose and
the citric acid cycle and decreased Mdh1 activity. Pnp is a ubiquitous intracellular enzyme and defective Pnp activity results in
fatal immune dysfunction (Toro et al., 2006). Energy production
and cellular immune function are closely related in mitochondria
(Burcham and Harman, 1991; Kasahara et al., 2011). In the present
study, we found that APAP down-regulated the expression of Mdh1
and Pnp, and dioscin reversed these changes. This regulation may
be related to the protective effect of dioscin against APAP-induced
liver damage.
Although these proteins have various biological functions, their
functions in liver injury may be mitochondrial. Thus, we think that
dioscin may trigger the mitochondrial pathway in its protective
effect against APAP-induced acute liver injury. Some of the proteins
related to the mitochondrial pathway were also investigated in the
present study.
p53 is known to promote apoptosis via either its transcriptional or non-transcriptional activity. In the former activity, p53
activates the transcription of pro-apoptotic Bcl-2 family members
(Weng et al., 2011). Bcl-2 prevents apoptotic formation factors such
as cytochrome c release from mitochondria, while Bax interacts
with mitochondrial voltage-dependent ion channels to mediate the
release of cytochrome c (Hassan et al., 2003). p53 can up-regulate
the expression of Bax and reduce the expression of Bcl-2 to complete the promotion of apoptosis (Weng et al., 2011). We measured
the expression of p53 after APAP treatment. APAP increased the levels of p53, likely as a consequence of activation of the mitochondrial
pathway. Dioscin prevented expression of p53, down-regulated
the expression of pro-apoptotic proteins, Bak and Bax, and upregulated the expression of anti-apoptotic proteins, Bcl-2 and Bid.
Our data provide evidence that dioscin suppresses APAP-induced
mitochondrial pathway activation, indicating this may be a relevant
mechanism for protection against cell death.
5. Conclusions
In conclusion, dioscin has a good protective effect against APAPinduced liver injury, and this protective action is related to the
regulation of mitochondrial function. Dioscin should be developed
as a new natural drug for the treatment of liver injury. Of course,
further research including clinical application, toxicology, mechanisms and drug targets require further investigation.
Conflict of interest
The authors declared that there are no conflicts of interest.
Acknowledgements
This work was supported by the Program for New Century Excellent Talents in University (no. NCET-11-1007) and the Program for
Liaoning Excellent Talents in University (no. 2009R15).
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.toxlet.2012.08.005.
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