TOXICOLOGICAL SCIENCES 79, 404 – 410 (2004)
DOI: 10.1093/toxsci/kfh120
Advance Access publication March 31, 2004
Changes in the Gene Expression Associated with Carbon
Tetrachloride-Induced Liver Fibrosis Persist after Cessation
of Dosing in Mice
Youchun Jiang,* Jie Liu,‡ Michael Waalkes,‡ and Y. James Kang* ,† , 1
*Department of Medicine, and †Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, Kentucky 40202; and
‡Inorganic Carcinogenesis Section, National Cancer Institute at NIEHS, Research Triangle Park, North Carolina 27709
Received December 9, 2003; accepted February 24, 2004
Recent studies have shown that gene expression profiles change
in the livers of animals treated acutely with toxic chemicals such
as carbon tetrachloride (CCl 4). This study was undertaken to
evaluate the changes in gene expression in mouse liver immediately after a long-term treatment with CCl 4 and possible effects of
treatment cessation on these changes. Adult 129/Sv pcJ mice were
treated twice a week with CCl 4 at 1 ml/kg in olive oil for 4 weeks.
Hepatic pathological changes observed in the CCl 4-treated mice
included necrosis, inflammation, and fibrosis, along with increased
serum alanine aminotransferase activities. Consistent with these
changes, expression of genes involved in cell death, cell proliferation, metabolism, DNA damage, and fibrogenesis were upregulated as detected by microarray analysis and confirmed by realtime RT-PCR. Four weeks after CCl 4 treatment cessation, the
pathological changes were recovered, with the exception of fibrosis, which was not completely reversed. Most of the gene expression profiles also returned to the control level; however, the fibrogenetic genes remained at a high level of expression. These results
demonstrate that changes in gene expression profile correlate with
pathological alterations in the liver in response to CCl 4 intoxication. Most of these changes are recoverable upon withdrawal of the
toxic insult. However, liver fibrosis is a prolonged change both in
gene expression and histopathological alterations.
Key Words: carbon tetrachloride; fibrosis; gene expression; immunohistochemistry.
Chemical-induced hepatotoxicity has been extensively studied in animal models, and the changes in biochemical pathways
in association with pathological progress in the liver under
toxic insults have been well documented (Armbrust et al.,
2002; Mukai et al., 2002; Simeonova et al., 2001; Stoyanovsky
and Cederbaum, 1999). Many studies using microarray technologies to characterize gene expression profiles in animals
exposed to toxicants have been undertaken recently (Bar1
To whom correspondence should be addressed at Department of Medicine,
University of Louisville School of Medicine, 511 S. Floyd St., DR 530,
Louisville, KY 40202. Fax: (502) 852-6904. E-mail: [email protected].
Toxicological Sciences vol. 79 no. 2 © Society of Toxicology 2004; all rights
reserved.
404
tosiewicz et al., 2001; Farr and Dunn, 1999; Liu et al., 2003;
Ruepp et al., 2000). There are thousands of genes that have
shown changes in their expression in response to toxic insults.
Although the significance of these changes has not been fully
understood, the information generated from microarray studies
indeed provides an alternative battery for evaluation of hepatic
responses to toxicants.
Carbon tetrachloride (CCl 4) is a well-investigated chemical,
and several microarray studies have been published describing
gene expression changes caused by acute CCl 4 toxicity (Bulera
et al., 2001; Harries et al., 2001). These gene expression
profiles have catalogued the molecular responses to acute CCl 4
toxicity and revealed the genetic basis of hepatic toxicity.
However, these acute studies have provided information regarding only acute phase responses and instant adaptation of
the liver to toxic insults. Changes in gene expression profiles in
response to a long-term exposure to toxicants have a fundamental impact on disease development. Equally important is
whether the changes in gene expression would be reversible, if
so to what extent, upon removal of the toxic insult.
To address these questions, we investigated changes in hepatic gene expression and hepatic pathology in response to
chronic treatment with CCl 4. Furthermore, we examined possible recovery of gene expression changes after removal of
CCl 4 and its correlation with the reversal of hepatic pathological changes. The data obtained indicate that there is a wide
range of changes in gene expression upon long-term CCl 4
treatment, and most of these changes can be reversed upon
treatment cessation. However, some changes were persistent.
In particular, CCl 4-induced up-regulation of genes involved in
liver fibrogenesis, which correlated well with the prolonged
fibrotic changes in the liver, remained after CCl 4 withdrawal.
MATERIALS AND METHODS
Animal experimental procedure. Adult 129/Sv pcJ mice (18 –20 g, 6 – 8
weeks of age) purchased from Jackson Laboratory were housed in cages with
a 12-h light/dark cycle (6 a.m. to 6 p.m.) and provided with rodent chow and
tap water ad libitum. The selection of this strain of mice was based upon the
increasing use of these mice for the study of liver disease using transgenic and
405
CCl 4 ALTERATION OF HEPATIC GENE EXPRESSION
knockout approaches and the highly demanded basic information regarding
hepatic toxic responses of these mice. All animals received humane care in
compliance with the institution’s guidelines, and animal procedures were
approved by the Institutional Animal Care and Use Committee, which is
certified by the American Association for Accreditation of Laboratory Animal
Care. Animals received an intraperitoneal injection of 1 ml/kg of CCl 4 (Sigma
Chemical, St. Louis, MO) in corn oil (1:4 ratio) or corn oil alone as control
twice weekly (Monday and Thursday) at about 10 a.m. for 4 weeks. There were
ten mice for each group. Five mice from each group were sacrificed 24 h after
the last injection, and the other five were sacrificed 4 weeks after the last
dosing. All the mice were subjected to an overnight fasting before they were
sacrificed.
Histopathological examination. The animals were anesthetized with avertin (0.4 mg/g), and blood was drawn from the dorsal vena cava, and sera were
obtained by centrifugation using a serum separator. The left lobe of the liver
was immediately removed and cross-sectioned into three equally sized pieces.
The middle piece was fixed in 10% buffered formalin, dehydrated in graded
ethylic alcohol, and embedded in paraffin. Sections at a thickness of 5 ␮m were
stained with hematoxylin/eosin (H/E) or picrosirius red. The H/E stained
sections were analyzed under light microscope for histopathological assessment. The others were stained with 0.1% Sirius red F3BA and 0.025% fast
green FCF (Junqueira et al., 1979) for fibrosis evaluation.
Immunohistochemical staining. Paraffin sections of 5 ␮m thick were
deparaffinized in xylene and dehydrated in alcohol. After treatment with 3%
(vol/vol) hydrogen peroxide in methanol for eliminating nonspecific reaction,
the samples were incubated overnight at 4°C with a 1:100 dilution of rabbit
polyclonal antibody against human TGF-␤1(Santa Cruz Biotechnology, Inc)
and a 1:300 dilution of rabbit anti-mouse collagen I (Chemicon International,
Temecula, CA). After incubation with the avidin-biotin complex, the antibody
labeling was visualized with diaminobenzidine and photographed using a light
microscope.
Measurement of serum alanine aminotransferase (ALT). Serum ALT
levels were measured using the sera obtained as described above by a standard
spectrometrically enzymatic method using a commercial kit (505 AST/GOT
kit, Sigma chemicals, St Louis MO).
Microarray analysis. The left lobe of the liver, with the exception of the
piece used for histopathological examination, from each of the five mice
described above was individually processed for RNA isolation. The total RNA
was isolated with Trizol威 Reagent (Invitrogen, Carlsbad, CA) and purified
with RNeasy columns (Qiagen, Valencia, CA). After the purification, aliquots
of the RNA samples from each of the five mice were pooled. The pooled RNA
sample from the five mice was used for microarray analysis in order to
eliminate the individual variability of changes within the group; however,
individual RNA samples were used for the real-time RT-PCR analysis. A total
amount of 2 to 5 ␮g of the pooled RNA was converted to ␣- 32P-dATP-labelled
cDNA probe using a Moloney murine leukemia virus reverse transcriptase and
the Atlas custom array specific cDNA synthesis primer mix (560 genes) and
purified with Nucleospin columns (Clontech). The membranes were prehybridized with Expresshyb (Clontech) for 60 min at 68°C and then hybridized
with labeled probes overnight. Hybridizations were performed in triplicate.
The membranes were washed four times with 2⫻ standard saline citrate (SSC)
containing 1% sodium dodecyl sulfate for 30 min followed by 2 washes with
0.1⫻ SSC containing 0.5% sodium dodecyl sulfate. The membranes were then
exposed to a Molecular Dynamics Phosphoimage Screen (Sunnyvale, CA).
The images were quantified densitomertrically by using Atlas Images v2.0
software (Clontech, Palo Alto, CA). The gene expression intensities were first
corrected for the external background and then globally normalized with the
sum of all genes on the array.
Real-time reverse transcription polymerase chain reaction analysis (realtime RT-PCR). In order to confirm changes in the gene expression detected
by the microarray analysis, real-time RT-PCR was performed to quantify the
selected genes. The total RNA isolated from each mouse liver was reverse
transcribed with the Moloney murine leukemia virus reverse transcriptase and
oligo-dT primers. The forward and reverse primers were designed using Primer
Express Software (Applied Biosystems, Foster City, CA). The SYBR green
DNA PCR kits (Applied Biosystems) were used for real-time RT-PCR analysis. The relative differences of gene expression among groups were evaluated
using cycle time values and expressed as relatively increases or decreases,
setting the values obtained from the wild-type mice treated with oil for 4 weeks
as 100%. Assuming that the cycle time value is reflective of the initial starting
copy and that there is 100% efficiency, a difference of one cycle is calculated
from each gene’s standard curve.
Statistical analysis. ALT data were expressed as mean ⫾ SD. For microarray, mean ⫾ SE values of three hybridizations were calculated. Statistical
differences between groups were determined by the paired Student t test.
Otherwise, data were analyzed using one-way ANOVA, followed by Duncan’s
multiple range tests. Differences were considered significant when p ⬍ 0.05.
RESULTS
Hepatotoxicity Induced by CCl 4
After the mice were treated with CCl 4 for 4 weeks, serum
ALT activity was increased significantly compared with untreated controls (250 ⫾ 45 vs. 7.8 ⫾ 5.6 Units/ml). Typical
necrotic changes in centrilobular areas were identified through
histopathological analysis (Fig. 1). The infiltrates of inflammatory cells were dominant in centrilobular areas. The picrosirius
red staining showed that fibrotic septa were formed after CCl 4
treatment for 4 weeks. Serum ALT activity was decreased to
the level of controls 4 weeks after the cessation of the CCl 4
treatment, and necrosis in the liver disappeared. However,
fibrotic changes were still observed.
Gene Expression Changes
The microarray analysis revealed significant changes in liver
gene expression induced by CCl 4. From the 560 genes present
on the array membranes, genes that displayed either greater
than or equal to a two-fold up- or down-regulation or p ⬍ 0.05
when compared to control group were selected for further
analysis. There were 21 genes whose expression was downregulated and 150 genes that were upregulated at the end of the
treatment with CCl 4 for 4 weeks. The majority of the downregulated genes were those involved in metabolism, and the
upregulated genes included those involved in cell proliferation,
cell death, oxidative stress, DNA damage, and extracellular
matrix regulation (Table 1). The selected genes are showed in
Table 1. After an additional 4 weeks in the absence of CCl 4,
more than 80% of the downregulated gene expressions were
corrected to the level of controls, but the expression of alcohol
sulfotransferase 1 and phenol/aryl form sulfotransferase (MSTP1) remained at a lower level compared with the control
(Table 1).
Of note, genes involved in cell proliferation were upregulated along with the genes involved in apoptosis (Table 1),
such as proto-oncogenes, cytokines, cyclins, and kinases.
There were more genes that are involved in cell proliferation
showing alterations in expression than apoptosis genes. After
the removal of CCl 4 for 4 weeks the expression of more than
406
JIANG ET AL.
genes, including procollagens, metalloproteinases, intergrin,
vimentin, and extracellular signal-regulated kinase 5, remained
at the high level of expression 4 weeks after the cessation of the
CCl 4 treatment.
Real-Time RT-PCR Analysis
Real-time RT-PCR was performed to confirm altered gene
expressions observed in the microarray analysis, as well as to
detect changes in the expression of some other genes that were
not included in the microarray analysis. The relative differences among groups were expressed as relative increases or
decreases to that of the control group treated with oil for 4
weeks, which was referred to as 100%. As shown in Table 2,
the results obtained from real-time RT-PCR were consistent
with those of the microarray analysis. The most significant
changes in gene expression were of genes involved in extracellular matrix regulation. All of the selected matrix genes
were upregulated greatly after the CCl 4 treatment for 4 weeks,
and most of these gene expressions remained at the high levels
after CCl 4 withdrawal for 4 weeks. Also in agreement with
results of the microarray analysis, c-myc, c-jun, cyclin D1,
p21, and cytokines were upregulated more than two times in
response to the treatment with CCl 4 for 4 weeks. Some of these
genes still had a higher level of expression 4 weeks after the
cessation of the CCl 4 treatment. Certain stress/DNA damage
genes also showed increased expression after CCl 4 treatment
for 4 weeks and decreased to the level of controls after CCl 4
withdrawal for 4 weeks, with the exception of GADD153. The
CYP2E1 gene showed a two-times decreased expression after
CCl 4 treatment for 4 weeks and regained its normal expression
level after cessation of CCl 4 treatment for 4 weeks.
Immunohistochemical Analysis of Fibrogenic Proteins
FIG. 1. Histopathological changes in the liver treated with CCl4 for 4
weeks and the recovery after cessation of the treatment for 4 weeks. A–D, the
tissue sections were stained with hematoxylin and eosin (H/E); E–H, the
tissues were stained with picrosirius red. A and E, olive oil treated for 4 weeks
as control; B and F, CCl 4 treated for 4 weeks; C and G, cessation with olive
oil treatment for 4 weeks; and D and H, cessation with CCl 4 treatment for 4
weeks. Arrow indicates fibrosis, and arrowhead necrotic changes. Original
magnification, ⫻160.
95% of the upregulated genes returned to the level of controls.
Two genes, Wee1/p87 (cdc2 tyrosine 15-kinase) and c-myc
proto-oncogene protein remained upregulated. Similarly, treatment with CCl 4 for 4 weeks caused up-regulation of genes
involved in the hepatic response to oxidative stress, and expression of most of these gene expressions returned to the level
of controls after the CCl 4 withdrawal, with the exceptions of
heat shock 105kD protein and Gadd45. One of the most dominant changes caused by CCl 4 treatment for 4 weeks was the
remarkably increased expression of the extracellular matrix
genes. Unlike other genes, the up-regulation of these matrix
The results obtained from both microarray and real-time
RT-PCR analyses showed that the most dominant change in
gene expression was the persistent up-regulation of the genes
involved in fibrogenesis. To further confirm this observation
and to link the molecular change with the observed pathological alterations, immunohistochemical detection of proteins
related to fibrosis was performed. The result shown in Figure 2
demonstrated the increased expression of TGF-␤ and collagen
type I proteins after CCl 4 treatment for 4 weeks. The TGF-␤
protein expression returned to the level of controls, but the
collagen type I expression remained high after CCl 4 withdrawal for 4 weeks.
DISCUSSION
This study showed that chronic treatment with CCl 4 caused
hepatic degenerative pathogenesis including cellular injury,
necrosis, and fibrosis. These pathological alterations, with the
exception of the fibrosis, were recoverable upon removal of
CCl 4. Microarray analysis revealed alterations in the expression
407
CCl 4 ALTERATION OF HEPATIC GENE EXPRESSION
TABLE 1
Changes in Hepatic Gene Expression After Treatment with CCl 4 for 4 Weeks and Their Recovery After Cessation of the
Treatment for 4 Weeks
Genes/proteins
Proliferation and cell death
Fas death domain-associated protein
c-myc proto-oncogene protein
embryonal carcinoma mRNA for cytokeratin 8
transforming growth factor beta 2
proliferating cell nuclear antigen; processivity factor
p21/Cip1/Waf1; cdk-inhibitor protein 1
hepatocyte nuclear factor 1 (HNF1)
Insulin I
c-Jun proto-oncogene
Ski proto-oncogene
Jun oncogenes
c-Src proto-oncogene
cyclin D1(o)
Wee1/p87; cdc2 tyrosine 15-kinase
caspase 2
Rb; pp105; Retinoblastoma susceptibility-associated
protein
Rac1; Ras-related C3 botulinum toxin substrate 1
interleukin beta 2
Metabolism
alcohol sulfotransferase 1
microsomal UDP-glucuronosyltransferase 2B5
precursor
cytochrome P450 2J5
phenol/aryl form sulfotransferase (M-STP1) gene
cytochrome P450, 2el
Glutathione s-transferase, microsomal
hepatic lipase
glucose-6-phosphate dehydrogenase
lysyl oxidase
DNA damage/stress
early growth response protein 1
heat shock 105kD protein
Gadd45
Gadd153 (growth arrest and DNA-damage-inducible
protein)
ERCC-1
ECM/cellular skeleton
matrix metalloproteinase 3
epithelial-cadherin
extracellular signal-regulated kinase 5
extracellular superoxide dismutase precursor (Cu-Zn)
matrix metalloproteinase 10
procollagen, type III, alpha 1
matrix metalloproteinase 2
matrix metalloproteinase 8
keratin, type II cytoskeletal 4
Intergrin alpha-3 precursor
procollagen, type I, alpha 2
Integrin beta 2
vimentin
thrombospondin 1
procollagen, type I, alpha 1
⌬ Fold
(p value)
Oil R
4 weeks
CCl 4 R
4 weeks
⌬ Fold
(p value)
1940 ⫾ 181 3019 ⫾ 239
2610 ⫾ 1025 5314 ⫾ 793
8849 ⫾ 3258 18109 ⫾ 1743
2896 ⫾ 225 5389 ⫾ 567
1392 ⫾ 579 3043 ⫾ 262
1130 ⫾ 159 2533 ⫾ 237
716 ⫾ 110 1650 ⫾ 262
694 ⫾ 313 1672 ⫾ 548
4011 ⫾ 460 9733 ⫾ 1478
4153 ⫾ 724 10645 ⫾ 1069
9637 ⫾ 1555 25659 ⫾ 5151
897 ⫾ 89
2450 ⫾ 172
4100 ⫾ 996 11954 ⫾ 1262
898 ⫾ 226 2677 ⫾ 396
372 ⫾ 66
1414 ⫾ 236
1.6 (0.012)
2.0
2.0 (0.025)
1.8 (0.009)
2.2 (0.019)
2.2 (0.002)
2.3 (0.024)
2.4
2.4 (0.017)
2.6 (0.002)
2.7 (0.040)
2.7 (0.000)
2.9 (0.002)
3.0 (0.010)
3.8 (0.008)
1778 ⫾ 143
1371 ⫾ 95
6866 ⫾ 1419
3713 ⫾ 507
1221 ⫾ 109
1079 ⫾ 339
921 ⫾ 41
664 ⫾ 6
4213 ⫾ 348
6710 ⫾ 636
8619 ⫾ 2041
749 ⫾ 123
3066 ⫾ 365
604 ⫾ 78
667 ⫾ 168
2335 ⫾ 266
3131 ⫾ 320
5225 ⫾ 642
4712 ⫾ 890
1126 ⫾ 247
1067 ⫾ 299
864 ⫾ 125
745 ⫾ 145
5560 ⫾ 956
5538 ⫾ 985
10464 ⫾ 2264
1216 ⫾ 96
1786 ⫾ 230
1580 ⫾ 126
825 ⫾ 254
1.3
2.3 (0.006)
0.8
0.7
0.9
1.0
0.9
1.1
1.3
0.8
1.2
1.6 (0.040)
0.6 (0.041)
2.6 (0.003)
1.2
281 ⫾ 110 1107 ⫾ 220
744135
3058 ⫾ 658
2855 ⫾ 254 13281 ⫾ 3042
3.9 (0.021)
4.1 (0.024)
4.7 (0.026)
271162
1178 ⫾ 179
2441 ⫾ 189
385151
974 ⫾ 51
4907 ⫾ 762
1.4
0.8
2.0 (0.035)
12912 ⫾ 4713 4637 ⫾ 1021
0.34 (0.06)
26696 ⫾ 4658
3789 ⫾ 822
0.14 (0.009)
28884 ⫾ 2205 15937 ⫾ 2398
35407 ⫾ 4077 14540 ⫾ 3621
10452 ⫾ 1124 5343 ⫾ 1395
49000 ⫾ 3571 25937 ⫾ 4257
46078 ⫾ 3375 15091 ⫾ 6111
9844 ⫾ 2336 4177 ⫾ 828
854 ⫾ 125 1883 ⫾ 254
484 ⫾ 232 1725 ⫾ 352
0.5 (0.006)
0.4 (0.006)
0.51 (0.03)
0.5 (0.005)
0.3 (0.005)
0.4 (0.028)
2.2 (0.015)
3.6 (0.032)
32389 ⫾ 2808
42080 ⫾ 5102
14330 ⫾ 2203
49175 ⫾ 4253
37617 ⫾ 3479
10546 ⫾ 936
921 ⫾ 293
495 ⫾ 36
38956 ⫾ 4550
37376 ⫾ 5567
4372 ⫾ 491
44900 ⫾ 5830
34428 ⫾ 8618
10809 ⫾ 2099
1010 ⫾ 205
848 ⫾ 170
1.20
0.81
0.31 (0.012)
0.91
0.92
1.02
1.1
1.7
7647 ⫾ 2175 15339 ⫾ 2235
2117 ⫾ 183 4357 ⫾ 546
1778 ⫾ 683 3795 ⫾ 276
2.0 (0.047)
2.1 (0.012)
2.1 (0.014)
6175 ⫾ 1120
1763 ⫾ 128
883 ⫾ 22
2097 ⫾ 165
2868 ⫾ 720
1355 ⫾ 147
0.3 (0.023)
1.6
1.5 (0.034)
3.5 (0.014)
3.6 (0.057)
497 ⫾ 31
745 ⫾ 79
747 ⫾ 88
503 ⫾ 133
1.5
0.7
2.0
2.0 (0.047)
2.1 (0.035)
2.1 (0.011)
2.2
2. (0.024)
2.6
3.4
3.4 (0.020)
3.5 (0.004)
4. (0.004)
4. (0.026)
4.7 (0.004)
4.9 (0.026)
9.6 (0.000)
647 ⫾ 184
2927 ⫾ 395
631 ⫾ 168
4652 ⫾ 230
525 ⫾ 90
924 ⫾ 87
663 ⫾ 131
343 ⫾ 27
682 ⫾ 213
1145 ⫾ 134
1527 ⫾ 156
2441 ⫾ 189
1920 ⫾ 262
710 ⫾ 336
1121 ⫾ 94
717 ⫾ 292
3356 ⫾ 256
1256 ⫾ 76
3967 ⫾ 237
758 ⫾ 131
3390 ⫾ 401
477 ⫾ 136
530 ⫾ 195
519 ⫾ 257
846 ⫾ 353
3258 ⫾ 409
4907 ⫾ 762
4824 ⫾ 461
657 ⫾ 324
3469 ⫾ 526
1.1
1.1
2.0 (0.027)
0.9
1.4
3.7 (0.004)
0.7
1.5
0.8
0.7
2.3 (0.010)
2.0 (0.035)
2.5 (0.005)
0.9
3.1 (0.012)
Oil
4 weeks
641 ⫾ 191
477 ⫾ 108
CCl 4
4 weeks
2230 ⫾ 386
1697 ⫾ 426
540 ⫾ 214 1107 ⫾ 327
1861 ⫾ 61
3813 ⫾ 664
636 ⫾ 132 1305 ⫾ 134
3266 ⫾ 348 6818 ⫾ 842
377 ⫾ 106
848 ⫾ 206
1638 ⫾ 189 4175 ⫾ 723
445 ⫾ 172 1165 ⫾ 346
522 ⫾ 42
1768 ⫾ 629
381 ⫾ 129 1301 ⫾ 241
806 ⫾ 223 2804 ⫾ 365
1534 ⫾ 138 7032 ⫾ 1110
2855 ⫾ 254 13281 ⫾ 3042
2227 ⫾ 87 10428 ⫾ 1612
584 ⫾ 120 2831 ⫾ 653
1016 ⫾ 64
9734 ⫾ 1024
Note: Data are mean ⫾ SE of three hybridizations from a pooled sample of five individual mouse livers.
408
JIANG ET AL.
TABLE 2
Real-Time RT-PCR Analysis of Selected Genes in the Liver of Mice Treated with CCl 4 for 4 Weeks and the Recovery After
Cessation of the Treatment for 4 Weeks
Genes
ECM-related genes
Procollagen, type I(␣1)
Procollagen, type III(␣1)
MMP-2
MMP-3
MMP-8
MMP-9
MMP-10
MMP-11
MMP-13
MMP-14
TIMP-1
TIMP-2
SMA
Cell proliferation/death-related genes
c-myc
c-jun
Cyclin D1
p21
TNF-a
TNF-R1
TGF-b
IL-6
IL-10
INF-g
Oxidative stress/DNA damage genes
ERCC 1
Gadd 153
Gadd 45
EGR 1
Metabolism CYP 2E1
Treated with CCl 4
for 4 weeks
Treated with oil
for 4 weeks
DFold for 4
weeks
Withdrawal CCl 4
for 4 weeks
Withdrawal oil
⌬ Fold
11.8
13.8
8.7
15.5
8.4
7.0
4.7
2.5
31.4
2.5
38.1
2.3
4.0
1.4
0.7
0.4
0.2
2.9
1.0
1.3
0.4
0.9
1.0
3.5
0.6
0.9
8.4
19.7
21.8
77.5
2.9
7.0
3.6
6.3
34.9
2.5
10.9
3.8
4.4
3.3
2.3
3.4
0.8
1.4
5.8
0.9
0.8
3.8
1.0
2.8
1.4
1.3
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
3.3
2.3
3.4
0.8
1.4
5.8
0.9
0.8
3.8
1.0
2.8
1.4
1.3
18.5
4.0
5.3
7.6
18.6
2.4
2.4
1.6
17.7
8.7
0.4
2.2
1.7
1.2
1.0
1.1
1.0
0.5
1.1
1.2
46.3
1.9
3.1
6.3
18.6
2.2
2.4
3.2
16.1
7.3
4.5
2.0
0.7
1.7
2.0
1.4
1.2
0.5
2.1
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
4.5
2.0
0.7
1.7
2.0
1.4
1.2
0.5
2.1
1.0
2.3
13.4
3.4
2.3
0.3
0.9
2.6
1.1
0.7
1.2
2.6
5.2
3.1
3.3
0.3
1.0
1.9
1.2
0.3
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.9
1.2
0.3
1.0
Note: Data are mean of five samples for each gene.
of many genes involved in acute phase response, metabolism,
cell death, cell proliferation, and fibrogenesis, and these observations were confirmed with real-time RT-PCR analysis. Most
of these gene expression changes were recovered after CCl 4
cessation; however, some important changes such as the upregulation of fibrogenesis genes remained. The persistent upregulation of several fibrogenesis genes is of particular interest,
because it correlates well with the persistent hepatic fibrosis
characteristic of CCl 4 exposure in mice.
Hepatotoxicity of CCl 4 has been a long-term focus of toxicological studies, and the pathological changes, along with
alterations in biochemical pathways induced by the hepatic
toxicant, have been well documented (Cabre et al., 2000;
Pietrangelo, 1996). Recent development in genomic technologies has led to new investigations into gene expression alterations caused by acute treatment with CCl 4 (Pietrangelo, 1996) .
Acute administration of CCl 4 to rats caused significant changes
in gene expression profiles (Fountoulakis et al., 2002; Waring
et al., 2001). The number of the genes affected increased with
the increased amount of the chemical administration. Some
genes appeared transiently affected, while others showed a
persistent alteration dependent upon the dose and time of
exposure. The most notable changes in CCl 4-treated animals
were the expression of genes involved in stress, DNA damage,
proliferation, and metabolic enzymes. Similar to the acute
administration, treatment with CCl 4 for 4 weeks, the majority
of the genes involved in the response to the toxicant were
upregulated. These included DNA damage and stress-related
genes such as GADD45, GADD153, and heat shock proteins,
cell proliferation and cell death-related genes such as c-myc,
Wee1/p87, Rb, PCNA, p21, cyclins, HNF-1, c-jun, and Fas
death domain-associated protein. Of note is that up-regulation
of cell proliferation and apoptosis-related genes were paralleled in response to CCl 4 insults. Most of the affected gene
expressions in these two categories were returned to untreated
control levels after CCl 4 withdrawal for 4 weeks. The excep-
CCl 4 ALTERATION OF HEPATIC GENE EXPRESSION
FIG. 2. Immunohistochemical staining of collagen type I (A–D) and
TGF-␤ (E–H) in the liver treated with CCl 4 for 4 weeks and the recovery after
cessation of the treatment with CCl 4 for 4 weeks. A and E, olive oil treated for
4 weeks as control; B and F, CCl 4 treated for 4 weeks; C and G, cessation with
olive oil treatment for 4 weeks; and D and H, cessation with CCl 4 treatment for
4 weeks. Arrow indicates collagen I staining, and arrowhead TGF-␤ staining.
Original magnification, ⫻160.
tions were that GADD153, c-myc, and Weel/p87 remained at
high levels of expression 4 weeks after cessation of CCl 4
treatment, indicating that the regulatory mechanisms that increased by CCl 4 treatment remained in an active state. These
genes might serve as markers to monitor the recover of liver
injury.
CCl 4 is metabolized to a peroxy radical by CYP2E1, resulting in increased lipid peroxidation (Johnston and Kroening,
1998; Wu and Cederbaum, 1996). Previous studies have shown
that acute treatment with CCl 4 resulted in down-regulation of
CYP2E1 (McGregor and Lang, 1996; Pietrangelo, 1996; War-
409
ing et al., 2001). In our study, the expression of CYP2E1 was
also decreased, together with CPY2J5 (p ⬍ 0.05). The most
interesting genes, including the alcohol sulfotransferase 1,
which oxidizes hydroxysteroid, and the phenol/aryl form sulfotransferase (M-STP1) gene, showed decreased expression
after treatment with CCl 4 for 4 weeks and did not showed any
recovery after cessation of CCl 4 treatment for 4 weeks. In
addition, the UDP-glucuronosyltransferase 1 gene, which plays
a major role in the detoxification and elimination of endogenous substrates, including bilirubin, bile acids, steroids, and
thyroid hormones, and exogenous compounds such as food
additives, therapeutic drugs and environmental pollutants, also
showed decreased expression after treatment with CCl 4 for 4
weeks. Both sulfotransferases and glucuronosyltransferases are
involved in phase II drug metabolism, and the inhibition of
their expression would alter the metabolic pathway of CCl 4 in
the liver. Interestingly, it has been shown that the activities of
these enzymes were inhibited by a high dose (1000 ␮l/kg) of
acute CCl 4 administration (Chardwick et al., 1988). Chronic
alcohol feeding also inhibited these two enzymes and generated mild fibrosis in the liver (Tadic et al., 2002). Therefore,
the CCl 4-suppressed expression of these enzymes might contribute to the hepatic fibrogenesis due to a metabolic shift.
In the development of liver disease, environmental stress
such as exposure to toxicants takes place over a long term, and
the accumulated effect on the biological system leads to the
degenerative process. Upon removal of the identified etiology,
the pathological changes may be reversible or continue to
develop to a persistent disease condition. Therefore, the molecular basis of the chronic responses of the liver to a long-term
exposure to CCl 4, in particular the reversibility of the changes
in the hepatic gene expression upon removal of the toxicant, is
of significant clinical relevance. In the present study, the most
dominant change in the gene expression was the up-regulation
of the fibrogenesis genes, and the changes were not recovered
4 weeks after the cessation of CCl 4 treatment. The molecular
changes relate well with the pathological alterations of the liver
(i.e., fibrosis was prolonged), although other pathological alterations were recovered after the CCl 4 withdrawal.
There are limitations in the present study. The most notable
is the selection of only 560 genes for the inclusion of this
study. Although the selection was based upon our current
understanding of hepatic toxicity of CCl 4, some important
responses in gene expression to this toxicant may be excluded.
Thus, the changes observed in the arbitrarily selective genes
only represent a partial response of the liver to the treatment
with CCl 4. Further studies are needed to fully explore the
response of hepatic gene expression to toxicants.
In summary, the results obtained from this study demonstrated for the first time that most changes in the hepatic gene
expression in response to a long-term treatment with CCl 4 were
recoverable, corresponding to the observed pathological
changes and their recovery. However, the genes involved in the
collagen deposition in the liver remain upregulated after the
410
JIANG ET AL.
cessation of the treatment with CCl 4, which would be highly
responsible for the persistent fibrosis in the liver.
ACKNOWLEDGMENTS
We thank Dr. Elaine Leslie and Dr. Yaxiong Xie for internal review of this
paper, and Dr. Zhanxiang Zhou for technical advice and assistance. This study
was supported in part by NIH grants HL59225 and HL63760. Y.J.K. is a
Distinguished University Scholar of the University of Louisville.
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