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http://het.sagepub.com/content/30/9/1369The online version of this article can be found at:

 DOI: 10.1177/0960327110391388

2011 30: 1369 originally published online 7 December 2010Hum Exp ToxicolWai Lim Ling, Kin Tung Tam and Jennifer Man-Fan Wan

Leo Lap Yan Wong, Sheung Tat Fan, Kwan Man, Wai-Hung Sit, Ping Ping Jiang, Irene Wing-Yan Jor, Carol Yee-Ki Lee,hepatotoxicity

Identification of liver proteins and their roles associated with carbon tetrachloride-induced  

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Identification of liver proteins andtheir roles associated with carbontetrachloride-induced hepatotoxicity

Leo Lap Yan Wong1, Sheung Tat Fan2, Kwan Man2,Wai-Hung Sit1, Ping Ping Jiang1, Irene Wing-Yan Jor1,Carol Yee-Ki Lee1, Wai Lim Ling1, Kin Tung Tam1, andJennifer Man-Fan Wan1

AbstractCarbon tetrachloride (CCl4) is a common hepatotoxin used in experimental models to elicit liver injury. Toidentify the proteins involved in CCl4-induced hepatotoxicity, two-dimensional gel electrophoresis wasemployed followed by mass spectrometry - mass spectrometry (MS/MS) to study the differentially expressedproteins during CCl4 exposure in the Fischer 344 rat liver proteome for 5 weeks. Ten spots with notablechanges between the Control and CCl4 groups were successfully identified. Among them, four proteins withsignificant up-regulation, namely calcium-binding protein 1, protein disulfide isomerase, mitochondrial alde-hyde dehydrogenase precursor, and, glutathione-S-transferase mu1 and six proteins with significant down-regulation, namely catechol-O-methyltransferase, hemoglobin-alpha-2-chain, hemopexin precursor, methio-nine sulfoxide reductase A, catalase and carbonic anhydrase 3, were identified. The data indicates that CCl4causes hepatotoxicity by depleting oxygen radical scavengers in the hepatocytes. In this rat model, we pro-filed hepatic proteome alterations in response to CCl4 intoxication. The findings should facilitate under-standing of the mechanism of CCl4-induced liver injury.

Keywordsreactive oxygen species, hepatotoxicity, liver, proteomics, carbon tetrachloride

Introduction

Carbon tetrachloride (CCl4)-induced hepatotoxicity is

the standard use of CCl4 as a liver toxin to induce liver

injury. It is a well-investigated experimental model

used to study the principles of hepatotoxic-induced

lipid peroxidation.1 CCl4 is a commercial solvent

used in machine cleaning and as a household agent for

the removal of stain in the past and was found to cause

intoxification.2 The hepatotoxicity was found to be

the result of inflammation initiated by oxidative

stress, free radicals and lipid peroxidation.3 CCl4-

induced hepatotoxicity mediates oxidative stress by

cytochrome P450 isoenzymes, resulting in trichloro-

peroyl free radical metabolites formation from a

chain of events involving plasma membrane in lipid

peroxidation.3 The free radical metabolites form

from the polyunsaturated fatty acids of the cell mem-

branes will adversely affect the structural component

of the membrane, resulting in alterations in fluidity

or permeability of membranes, cell energy processes

destruction and protein synthesis.4-7 Such mechan-

isms are believed to be the cause of liver injury

by CCl4.

Proteomics has gained increasing popularity in the

investigation of quantitative change in protein expres-

sion in biological systems.8,9 Such techniques provide

1 School of Biological Sciences, The University of Hong Kong,HKSAR, China2 Department of Surgery, The University of Hong Kong, HKSAR,China

Corresponding author:Jennifer Man-Fan Wan, The University of Hong Kong, School ofBiological Sciences, 5S-01 Kadoorie Biological Sciences Building,Pokfulam, Hong Kong SAR, ChinaEmail: jmfwan@hkusua.hku.hk

Human and Experimental Toxicology30(9) 1369–1381

ª The Author(s) 2010Reprints and permission:

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bioinformatics for obtaining a better understanding of

the disease pathogenesis. Differences in protein

expression have been recorded in liver cancer with the

use of proteomic analysis.10 In the current study, a

two-dimensional gel-based proteomic approach was

adopted to examine the liver proteome changes in

response to chronic treatment with CCl4.

Materials and methods

Animals

Adult male Fischer 344 rats (initial body weights ¼250�280 g; n ¼ 5 for each group; total n ¼ 10) from

our own breeding colony were used. The animals

were kept in a separate animal room, on a 12-h

light-dark cycle, at a room temperature 22�C and con-

stant humidity with access to standard laboratory

chow and water ad libitum. Animal care and treatment

were conducted and approved according to the guide-

lines of the Committee on the Use of Live Animals in

Teaching and Research, The University of Hong

Kong,11 and Department of Health, the Government

of Hong Kong, HKSAR.

Generation of liver injury model

All rats were allowed to acclimatize for a week before

the experiment. The rats were randomly assigned into

Control and CCl4 groups. The Control group received

subcutaneous injection with pure olive oil twice a

week for 5 weeks. The CCl4 group received subcuta-

neous injection with CCl4 (Merck KGaA, Darmstadt,

Germany) diluted 1:1 (v/v) in pure olive oil at a

dose of 0.2 mL/100 g of body weight twice a week for

5 weeks. This hepatotoxicity animal model was

described previously, with modification.12

Tissue collection

At the end of experiment, animals were anesthetized

with ether and sacrificed. Blood samples were taken

by cardiac puncture and the collected serum was used

for biochemical analysis. The liver tissue specimens

were removed immediately and approximately 1 cm

sections of the right lobe were harvested, snap-

frozen in liquid nitrogen and stored at –80�C for liver

enzyme assays and proteomic analysis.

Biochemical assays

Alanine aminotransferase (ALT) and g-glutamyl

transferase (g-GT) testing kits were purchased from

Biosystems S.A. (Barcelona, Spain). The serum levels

were measured spectrophotometrically using UA-

visible recording spectrophotometer (Shimadzu,

Japan) according to manufacturer’s instructions. The

level of reduced glutathione (GSH) was measured

by the method described by Akerboom et al.13 Briefly,

liver was homogenized in ice-cold perchloric acid

containing EDTA. The obtained acidic supernatant

was neutralized by potassium hydroxide containing

3-[N-morpholino] propanesulphonic acid (MOPS). To

measure GSH, the neutralized sample was mixed with

glyoxalase I and phosphate buffer (pH 6.5). The absor-

bance (240 nm) was measured exactly 10 minutes after

the addition of methylglyoxal. For the measurement in

the activities of glutathione-S-transferase (GST), cata-

lase (CAT) and glutathione peroxidase (GPx), the liver

was homogenized in ice-cold phosphate buffer (pH 7.4)

and the supernatant was obtained. The GST activity was

determined by measuring the rate of formation of conju-

gate of 1-chloro-2,4-dinitrobenzene (CDNB) according

to the procedure of Habig et al.14 CDNB-GSH

conjugate was measured at absorbance (340 nm) over

5 minutes. In the CAT activity assay, the supernatant

was reacted with H2O2, and the unconverted H2O2

reacted with H2SO4 and KMnO4 to form MnO2. The

absorbance (480 nm) was measured over 1 minute.15

The activity of GPx was determined by the method of

Paglia and Valentine,16 with modification. Briefly, the

supernatant was added to phosphate buffer (pH 7.0)

containing EDTA, GSH, sodium azide (NaN3),

NADPH and glutathione reductase. After addition of

cumnene hydroperoxide (CumOOH), the rate of oxida-

tion of NADPH was measured at absorbance (340 nm)

over 5 minutes.

Sample preparation for proteomic analysis

Protein extraction method has been described previ-

ously.8 The liver tissue samples were disrupted with

a tissue teaser (Biospec Products, Oklahoma, USA)

in a cocktail buffer (1 % Triton X-100, 25 mmol/L

HEPES, 150 mmol/L NaCl, 1 mmol/L EDTA diso-

dium salt, 1 mmol/L dithiothreitol [DTT]) with

added Protease Inhibitor Cocktail Set III (Bio-Rad,

California, USA). The superfluous salt in the super-

natant was removed by incubating with trichloroace-

tic acid (TCA)-acetone solution (20 % TCA, 20

mmol/L DTT in acetone) for 4 h at –40�C. The

protein pellet was obtained by centrifugation at

15,800 x g for 30 minutes at 4�C. Excess TCA was

removed by multiple washing with acetone

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containing 20 mmol/L DTT. The air-dried protein

pellet was resuspended in buffer (7 mol/L urea, 2

mol/L thiourea, 4% 3-[(3-cholamidopropyl)

dimethylamonio]-1-propanesulfonate, 100 mmol/L

DTT, 5% glycerol). The final protein solution was

stored at –80�C until further 2-DE analysis. Protein

concentration of each sample was measured by spec-

trophotometer using Bio-Rad protein microassay

based on the method of Bradford.

Two-dimensional gel electrophoresis andMS/MS analysis

The two-dimensional gel electrophoresis (2-DE)

procedures used here have been previously described

by us with modifications.9 A total of 10 gels (5 for

Control and 5 for CCl4) were conducted, with one gel

per animal from each of the time points. For the first

dimensional electrophoresis of proteins, a fixed

amount of 125 mg protein samples were loaded onto

an 18 cm ReadyStrip immobilized pH gradient (IPG)

Strips (pI 3-10 NL, Bio-Rad, California, USA). The

strips were rehydrated with 350 mL rehydration buf-

fer (9.5 mol/L urea, 2 % 3-[(3-cholamidopropyl)

dimethylamonio]-1-propanesulfonate, 0.28 % DTT,

0.5% IPG buffer, pH 3–10, 0.002% bromophenol

blue) before being applied onto the Ettan IPGphor III

isoelectric focusing (IEF) electrophoresis system

(GE Healthcare). The samples were rehydrated for

7 h before isoelectric focusing via the following

programs: (1) linear increase up to 500 V in 1 h; (2)

held at 500 V for 3 h; (3) linear increase up to

10,000 V in 3 h; (4) linear increase up to 10,000 V

in 3 h and (5) finally held at 10,000 V to reach a total

of 90,000 Vhs. Focused IPG gel strips were equili-

brated for 15 minutes in a solution (50 mmol/L

Tris-HCl, pH 8.8, 6 mol/L urea, 30 % glycerol, 2 %SDS, containing 20 mmol/L DTT) followed by incu-

bation with the same buffer containing 20 mmol/L

iodoacetamide for another 15 minutes. Equilibrated

gel strips were then placed onto a 1.0-mm-thick

12.5 % polyacrylamide gels and SDS-PAGE was car-

ried out at a constant current of 30 mA for 30 minutes

followed by a 60-mA current for the rest of the

analysis in a PROTEAN xi II cell (Bio-Rad,). After

electrophoresis, gels were fixed in fixation

solution (10% ethanol, 7% acetic acid in water) for

2 h before staining with SYPRO Ruby Protein

stain (Bio-Rad), according to the manufacturer’s

guideline.

Image acquisition and analysis

Image analysis and protein identification. The Molecular

Imager PharosFX Plus System (Bio-Rad) was used

to scan stained 2-DE gels. PDQuest 8.0 for Windows

(Bio-Rad) software was used for matching and ana-

lyzing protein spots on the 2-DE gels, with the func-

tion on background subtraction, spots detection and

volume normalization. Detected spots was normal-

ized and assigned with a number referenced by a

‘virtual gel’ or a reference gel (the best gel of the

10 gels) automatically. Any under-detected spots

were manually assigned with the number according

to the reference gel by the researcher. The expression

level, expressed as percentage volume (% vol), was

exported for statistical analysis. Protein spots with

differential expression between Control group and

CCl4 group (p < 0.05) were selected for protein

identification.

Protein identification by MS/MS

Spots with differential expression (p < 0.05) between

the Control and CCl4 groups were sent to the Genome

Research Centre (The University of Hong Kong,

Hong Kong) for protein identification. The proteins

were digested with trypsin and applied to matrix-

assisted laser desorption ionization-time of flight/time

of flight (MALDI-TOF/TOF MS) analysis on 4800

MALDI TOF/TOF Analyzer (Applied Biosystems,

CA, USA) for analysis. The match between the

experimental data and mass values calculated from

a candidate protein was carried out by Mascot peptide

mass fingerprinting, a powerful search engine that

uses MS data to identify proteins from www.matrixs-

cience.com against the NCBI database with taxonomy

limited to Rattus norvegicus (Rats). Mascot reported

the molecular weight search (MOWSE) score, which

is calculated by –10� Log10 (p), where p is the prob-

ability that the observed match is a random event. p is

limited by the size of the sequence database being

searched (limited by taxonomy), the conditions and

the settings of trypsin digestion. Each calculated

value that falls within a given mass tolerance of an

experimental value counts as a match. The accepted

threshold shows an event is significant if it would

be expected to occur at random with a frequency of

<5%. In this study, a protein match with a score >64

was regarded as significant.17 The Mascot also gener-

ated values of sequence coverage, which is the per-

centage of trypsin-derived peptides accounting for

the whole sequence of the target protein. The apparent

Wong L L Y et al. 1371

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isoelectric point and relative molecular mass of

proteins are calculated according to their position on

the gels and confirmed with the searched data.

Western blotting analysis

Liver protein extract was mixed with sample buffer

(62.5 mmol/L Tris, pH 6.8, 25% glycerol, 2% SDS,

350 mmol/L DTT, 0.01% bromophenol blue). Thirty

mg protein of each sample was applied for electro-

phoresis on 12.5% SDS-PAGE gels with constant

voltage (125 V) and transferred to polyvinylidene

diflouride membrane. The membranes were incubated

with primary antibodies and subsequently with sec-

ondary antibodies as listed below: rabbit polyclonal

against Catalase (CAT) or protein disulphide isomer-

ase (PDI; secondary antibody: goat anti-rabbit conju-

gated with horseradish peroxidase); goat polyclonal

against catechol-O-methyltransferase (COMT) or

glutathione-S-transferase mu (GSTM1; secondary

antibody: rabbit anti-goat conjugated with horserad-

ish peroxidase); chicken polyclonal against calcium

binding protein 1 (CABP1; secondary antibody: goat

anti-chicken conjugated with horseradish peroxi-

dase); mouse monoclonal against mitochondrial alde-

hyde dehydrogenase 2 (ALDH2) or b-actin

(secondary antibody: goat anti-mouse conjugated

with horseradish peroxidase). All primary antibodies

were purchased from Abcam, Cambridge, UK, except

ALDH2, which was purchased from Santa Cruz

Biotechnology, USA. All secondary antibodies were

purchased from Bio-Rad. The intensity change of

protein bands was accessed using Quantity One soft-

ware (Bio-Rad). The relative molecular weight of

each protein band was estimated with molecular

markers (Precision Plus Protein Standards Dual

Color, Bio-Rad) and was detected using ECL Western

blotting detection kit (Amersham, Arlington Heights,

Illinois, USA).

Statistical analysis

All data were analyzed by SPSS 11.5 software and

reported as means + SEM using one-way ANOVA.

Student’s t-test was used for between-groups compar-

ison. p Values of 0.05 or less are considered statisti-

cally significant.

Results

Body weight and liver weight

At the end of experiment, the mean body weight of the

rats in the CCl4 group was 287.4 + 19.96 g and in the

Control group was 312.4 + 9.6 g, with no significant

difference (Figure 1A). In contrast, the mean liver

weight in the CCl4 group (22.9 + 0.72 g) was signif-

icantly heavier than those in the Control group (16.55

+ 0.32 g; p < 0.001; Figure 1B).

Figure 1. Effects of carbon tetrachloride (CCl4) on mean body weight (g) (A) and mean liver weight (g) (B). Male F344 ratswere subcutaneously injected with or without CCl4 (0.2 mL/100g/twice a week) for 5 weeks. Values are mean + SEM forfive rats per group. ***p < 0.001 compared with the control group.

1372 Human and Experimental Toxicology 30(9)

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Biochemical assay of liver function CCl4-inducedhepatotoxicity

The data revealed that the administration of CCl4compared to Control significantly increased the serum

activity of ALT by 40-fold (2798.2 + 193.9 U/L in

CCl4 group and 65.02 + 3.2 U/L in Control group;

p < 0.001; Figure 2A) and g-GT by 20-fold (6.62 +0.67 U/L in CCl4 group and 0.29 + 0.08 U/L in

Control group; p < 0.001; Figure 2B).

GSH and GST assays on glutathione metabolismin CCl4-induced hepatotoxicity

Subcutaneous administration of CCl4 significantly

depleted hepatic GSH concentration by 74% from

normal rats 6.68 + 0.05 mmol/g to 1.75 + 0.33

mmol/g (p < 0.001; Figure 3A) and significantly ele-

vated hepatic GST activity by 62% from normal rats

of 0.336 + 0.064 mmole CDNB/min/mg to 0.895 +0.124 mmole CDNB/min/mg (p < 0.01; Figure 3B)

when CCl4 was administered.

CAT and Gpx activities in CCl4-inducedhepatotoxicity

CCl4 significantly reduced hepatic CAT activity

by 74% from normal rats of 5.14 + 0.21 mmole

H2O2/min/mg to 1.34 + 0.18 mmole H2O2/min/mg

(p < 0.001; Figure 3C) and the level of hepatic

Gpx was significantly reduced by 31% from normal

rats of 0.136 + 0.008 mmole NADPH/min/mg to

0.094 + 0.008 mmole NADPH/min/mg (p < 0.001;

Figure 3D) when CCl4 was administered.

Liver proteome profile and proteinidentification

There were approximately 500 well-resolved protein

spots detected on each 2-DE gels. Ten spots with

notable changes between the Control and CCl4 groups

were successfully identified (Figure 4). The subcellu-

lar locations were examined and the basic information

on the identified proteins with a lower or higher

expression level (p < 0.05) in the CCl4 group was

compared with Control (Table 1). The proteins with

increased expression were calcium binding protein 1

(CABP1; spot 407), protein disulfide isomerase (PDI;

spot 502), mitochondrial aldehyde dehydrogenase

precursor (ALDH2; spot 2118), and glutathione-S-

transferase mu 1 (GSTM1; spot 8129). The proteins

with decreased expression were catechol-O-methyl-

transferase (COMT; spot 17), hemoglobin alpha 2

chain (HBa2; spot 36), hemopexin precursor (HPX;

spot 2611), methionine sulfoxide reductase A

Figure 2. Serum biochemical assays of alanine aminotransferase (ALT) (A) and g-glutamyl transferase (g-GT) (B) liverfunction in response to carbon tetrachloride (CCl4)-induced hepatotoxicity. Rats were treated with or without CCl4 for5 weeks. Serum samples were assayed for the activities of ALT and g-GT. Values are mean + SEM for five rats per group.***p < 0.001 compared with the control group.

Wong L L Y et al. 1373

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(MSRA; spot 5113), catalase (CAT; spot 6504) and

carbonic anhydrase 3 (CAR3; spot 7111).

Western blotting confirmation

The results are similar to those of the 2-DE proteo-

mic analysis (Table 1), showing both COMT and

CAT were reduced (p < 0.05), whereas GSTM1,

CABP1, PDI and ALDH2 were increased (p <

0.05) in the CCl4 group compared with the Control

group (Figure 5).

Discussion

In this study, we demonstrated that the injection of

CCl4 for 5 weeks in rats led to a marked elevation

in the levels of serum ALT and g-GT. Both the cyto-

plasmic ALT and g-GT released into the circulation

after cellular damage have been considered as hepatic

damage biomarkers in the diagnosis of liver injury.18

Our data (Figure 3) indicates the presence of a sup-

pression of the antioxidant system in the liver of rats

treated with CCl4 since GSH is an antioxidant that

helps protect cells from reactive oxygen species

(ROS), such as free radicals and peroxides. In the

liver, CCl4 is metabolized by cytochrome P450 in the

endoplasmic reticulum to produce the highly reactive

trichloromethyl radical, which is further converted to

the peroxytrichloromethyl radical. This free radical

can extract hydrogen from different molecules, thus

initiating the oxidation of lipids, proteins and DNA

in liver injuries.19

In order to construct a database to improve our

understanding of hepatotoxicity and of potential diag-

nostic or therapeutic protein target discovery, we

Figure 3. Liver enzyme assays of reduced glutathione (GSH) (A), glutathione-S-transferase (GST) (B), catalase (CAT) (C)and glutathione peroxidase (GPx) (D) in response to carbon tetrachloride (CCl4)-induced hepatotoxicity. Rats weretreated with or without CCl4 for 5 weeks. Liver homogenate samples were assayed for the activities of GSH, GST, CATand Gpx. Values are mean + SEM for five rats per group. **, *** p < 0.01, 0.001 as compared with the Control group,respectively.

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applied a comprehensive analysis of proteins

associated with the hepatotoxicity induced by chronic

exposure of rats to CCl4 by using a combination of

two-dimensional electrophoresis (2-DE) and mass

spectrometry. The 2-DE proteomic technique is use-

ful for systematic analyses of global protein abun-

dance profiles of normal and diseased tissues, often

providing novel insights into the pathogenic mechan-

isms of human disease as well as disease-associated

targets. In the present study, we have successfully

identified 10 proteins from the NCBI database, and

their importance in the pathophysiology of CCl4-

induced hepatotoxicity is discussed below under the

following clusters: detoxification and antioxidation,

hormone regulation, amino acid metabolism, protein

transportation and protein synthesis and calcium

homeostasis (Table 1).

Detoxification and antioxidation

Carbonic anhydrase 3 (CAR3) is a member of the car-

bonic anhydrase family of zinc metalloenzymes.

Apart from aiding the transport of carbon dioxide out

of tissues, it may also serve as an oxygen radical sca-

venger to protect hepatocytes from oxidation damage.

The enzyme contains two reactive sulfhydryl groups

(Cys 181 and Cys 186) that readily form disulfide lin-

kages with GSH, a process termed S-glutathiolation,

under conditions of oxidative stress.20 Thus, depletion

of CAR3 could provoke the oxidative process from

decreasing the production of disulfide linkages

formed induced by CCl4. Furthermore, CAR3 has

been shown to protect hepatocytes from hydrogen

peroxide-induced apoptosis.21-27 The reduced abun-

dance of this protein in the present study (fivefold

decrease) may have allowed the production of free

radicals in the hepatocytes. This may be a mechanism

by which the rat hepatocytes were damaged by CCl4(Figure 6).

Glutathione-S-transferase mu1 (GSTM1) is an

antioxidant enzyme that has a protective effect against

oxidative stress28 and a capacity to detoxify endogen-

ous compounds, such as peroxidised lipids, by con-

verting GSH to GSH-free radical conjugate.29-32

It has been proposed that human subjects lacking

GSTM1 would be more susceptible to the genotoxic

effects of carcinogenic epoxides.33 In this study, the

hepatic GST activity was significantly increased by

62% (Figure 3B) and the result of proteomics in

GSTM1 was significantly up-regulated (12-fold

increase) when treated with CCl4. These results indi-

cated that the decrease in GSH was related to an

increase in the overall production of GST, which sub-

sequently catalyzes formation of GSH-free radical

conjugate (Figure 6).

Mitochondrial ALDH2 was significantly increased

in CCl4-treated rats. This protein is responsible for

eliminating xenobiotic aldehydes and toxic biogenic

Figure 4. Representative 2-DE gels of a rat liver proteome map, with or without the administration of carbon tetrachlor-ide (CCl4) for 5 weeks (n ¼ 5 for each groups). Immobilized pH gradient (IPG) strips (18 cm pH 3�10) were used forisoelectric focusing (IEF), followed by SDS-PAGE (12.5% polyacrylamide). The gels were visualized using SYPRO Ruby pro-tein staining and the differentially expressed proteins were numbered.

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Tab

le1.

Rat

liver

pro

tein

sex

hib

itin

gdiff

eren

tial

expre

ssio

nw

ith

or

without

the

adm

inis

trat

ion

ofca

rbon

tetr

achlo

ride

(CC

l 4)

Spot

no

aPro

tein

Gen

Info

iden

tifie

rbM

OW

SEsc

ore

c

Expre

ssio

nquan

tity

inco

ntr

olra

td

(�10

4)

Expre

ssio

nquan

tity

inC

Cl 4

rats

d

(�10

4)

Contr

ol

vs.C

Cl 4

appro

xim

ate

fold

-chan

ges

pe

pIf

Mr

(kD

a)f

Subce

llula

rlo

cation

Funct

ions

Up-r

egula

ted

pro

tein

s407

Cal

cium

bin

din

gpro

tein

1(R

attu

sno

rveg

icus

)gi

|488838

261

100.2

4+

27.5

5264.9

7+

37.0

42.6

40.0

13

4.9

547.6

Cyt

opla

smC

alci

um

hom

eost

asis

502

Pro

tein

dis

ulfi

de-

isom

eras

e(R

attu

sno

rveg

icus

)

gi|1

29731

611

490.2

6+

117.1

61302.7

0+

127.0

02.6

60.0

03

4.8

257.3

Endopla

smic

reticu

lum

Pro

tein

synth

esis

2118

Mitoch

ondri

alal

deh

yde

deh

ydro

genas

epre

-cu

rsor

(Rat

tus

norv

egic

us)

gi|4

5737868

180

0.0

01+

0.0

049.4

5+

2.3

749.4

50.0

01

7.6

356.1

Mitoch

ondri

on,

mitoch

ondri

alm

atri

x

Det

oxifi

cation

8129

Glu

tath

ione

S-tr

ansf

eras

e,m

u1

(Rat

tus

norv

egic

us)

gi|8

393502

157

36.7

1+

13.4

8429.3

8+

61.5

411.7

00.0

49

8.2

726.1

Cyt

opla

smD

etoxifi

cation/

Antioxid

atio

n

Dow

n-r

egula

ted

pro

tein

s17

Cat

echol-O

-m

ethyl

tran

sfer

ase

(Rat

tus

norv

egic

us)

gi|6

978681

96

154.9

4+

28.5

753.8

0+

16.7

2�

2.8

80.0

26

5.4

129.8

Cyt

opla

smH

orm

one

regu

lato

r

36

Hem

ogl

obin

alpha

2ch

ain

(Rat

tus

norv

egic

us)

gi|6

0678292

311

139.2

4+

29.9

44.8

4+

2.0

7�

28.7

70.0

04

8.4

515.4

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1376

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agents34-36 and also has a major role in acetaldehyde

detoxification.37 It is believed that it may play a protec-

tive role in the mechanism by which hepatocytes com-

bat sudden increases in aldehyde products. The 49-fold

increase of ALDH2 is a strong indication of the pres-

ence of increased lipid peroxides in the CCl4-treated

liver. In this pathway, the intermediate structures can

be toxic and health problems may arise when those

intermediates cannot be cleared38 (Figure 6).

Catalase (CAT) is a ROS scavenger responsible for

the breakdown of H2O2. The reduced expression of

catalase and glutathione peroxidase (GPx) in hepato-

cytes is generally correlated to the decrease in the

GSH/GSSG ratio and increase in lipid peroxidation.39

In the present study, the hepatic CAT activities was

significantly decreased by 74% (Figure 3C) and there

was a reduced expression of hepatic GPx activity by

31% when CCl4 was administered (Figure 3D). We

conclude that the fourfold decrease in CAT in liver

proteome could lead to an increase in hepatocyte lipid

peroxidation in the animals (Figure 6).

Amino acid metabolism

The present study shows that CCl4 damage to the rat

liver led to a decrease in the abundance of methionine

Figure 5. Western blot analysis of liver proteins, catechol-O-methyltransferase (COMT) (A), catalase (CAT) (B),glutathione-S-transferase mu (GSTM1) (C), protein disulphide isomerase (PDI) (D), calcium binding protein 1 (CABP1)(E) and mitochondrial aldehyde dehydrogenase 2 (ALDH2) (F). Values are mean + SEM; n ¼ 5 in each groups. *,*** p < 0.05, 0.001 as compared with the Control group, respectively.

Wong L L Y et al. 1377

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sulfoxide reductase A (MSRA), which is responsible

for catalyzing the thioredoxin-dependent reduction

of free and protein-bound methionine sulfoxide

[Met(O)] residues to methionine. According to previ-

ous studies, it was indicated that mouse lacking the

MSRA gene exhibited enhanced sensitivity to oxida-

tive stress when compared with the wild type. It is

believed that MSRA may possibly play an important

role as an antioxidant in mammals.40 The 13-fold

depletion of MSRA found in this study may have

resulted in a decrease in the reduction of Met(O) resi-

dues to methionine, a ROS scavenger, which could

increase the susceptibility of the rat liver to oxidative

stress imposed by ROS (Figure 6).

Hormone regulator

A threefold depletion of catechol-O-methyl transfer-

ase (COMT) was found in this study. COMT is

responsible for converting catechol to guaninol and

has been found to serve as a mechanism of preserving

S-adenosylmethionine (SAM) in hepatocytes.21,41

Moreover, COMT inhibitor application for the treat-

ment of Parkinson’s disease has been shown to cause

potential hepatotoxicity.42 It is speculated that

decreased availability of methionine would lead to the

shortage of SAM, a substrate of COMT, causing

reduced level of S-adenosylhomocysteine (SAH). As

a result, the reduced level of SAH could cause

lowered biosynthesis of cysteine and hence decrease

of GSH production (Figure 6).

Protein transportation and protein synthesis

We observed that CCl4 treatment caused twofold

decrease in Hemopexin precursor (HPX) and 29-

fold decrease in Hemoglobin alpha 2 chain (HBa2).

Since both of them are transporters of heme to liver

parenchymal cells and active in heme scavenging,43

the reduction of HPX and HBa2 could result

increased level of free heme, which is highly toxic

as it promotes the generation of ROS and thus pro-

moting oxidative damage.44,45 Moreover, heme is

a positive regulator of cytochrome P450 gene

transcription.46 It is believed that free radicals

(CCl3- and CCl3OO-) generated by the cytochrome

P450-dependent detoxification step could induce

liver injury by lipid peroxidation (Figure 6). The

increase in cytochrome P450, however, cannot be

shown by proteomic analysis in the present study,

due to the fact that cytochrome P450 proteins are

endoplasmic reticulum membrane proteins with a

very high isoelectric point (pI), which makes 2-DE

analysis difficult.34,47,48

The PDI, which exhibited a threefold increase in

the present study, is a multifunctional protein. PDI

Figure 6. Proposed inter-relationships of the differentially expressed liver proteins in carbon tetrachloride (CCl4)-induced hepatotoxicity

1378 Human and Experimental Toxicology 30(9)

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plays important roles in corrective protein folding and

as a cellular chaperone.49 It is also involved in the

maturation of collagen helix, which is a landmark of

liver fibrosis which is increased in liver injury. The

intrinsic function of this protein, however, is believed

to be the rearrangement of disulfide bridges and the

catalysis of oxidation, reduction and corrective disul-

fide isomerization so as to prevent unnecessary aggre-

gation.49 Under the oxidative stress induced by CCl4consumption, proteins are highly susceptible to

cysteine oxidation and the incorrect formation of disul-

fide bridges, leading to detrimental change in the

3-dimensional structure of the protein and aggrega-

tion.49 Moreover, it is hypothesized that under signifi-

cant oxidative stress in the injured hepatocytes, the

increased abundance of PDI production is to compen-

sate for the demand of corrective disulfide isomeriza-

tion, which is needed to stabilize the proteins

susceptible to aggregation (Figure 6).

Calcium homeostasis

CABP1 is a high-affinity calcium-binding glycopro-

tein of the rat liver endoplasmic reticulum.50 Studies

have indicated the cytosolic calcium level is elevated

after exposure to hepatotoxins such as CCl4. This ele-

vated cytosolic Ca2þ is believed to be the result of an

activation of calcium-releasing channels.51-53 The

threefold increase in CABP1 found in the present

study supports the fact that CCl4 metabolism may

play a role in the activation of calcium release into the

cytosol (Figure 6). The high levels of calcium in the

cytosol activate calcium-dependant proteases that

exacerbate membrane damage. The increase in intra-

cellular calcium can activate endonucleases that

inflict chromosomal damage that in turn contributes

to cell death and cancer.54

In summary, CCl4 is a hepatotoxicant that gener-

ates ROS and initiates lipid peroxidation and induces

significant liver injury in the rats. We propose that the

hepatotoxicity mechanism by which CCl4 induced

liver injury in the rats is mostly due to the suppression

of the antioxidant and detoxification system. Also, in

this study, we showed that proteomic technology can

serve as a powerful tool for the rapid identification

and confirmation of proteins with significant roles

in the pathogenesis of liver injury during toxic chem-

ical exposure. The liver proteomic analysis approach

hastened the discovery of significant proteins that

are involved in the pathogenesis of the liver injury

induced by the hepatotoxicant. MS/MS-based

sequencing of peptides derived from these

differentially expressed proteins has helped identify

both protein markers that had previously been cited

as possible indicators as well as potentially novel

associations with hepatic effects for proteins already

established by other investigations. The present data

facilitate not only the molecular mechanistic investi-

gation of CCl4-induced liver injury but also provide

new direction in the study of disease pathogenesis.

Funding

This work was supported by the grants of the Liver Strate-

gic Research Grant by The University of Hong Kong.

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