* 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
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ABSTRACT |
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Key Words: carbon tetrachloride; fibrosis; gene expression; immunohistochemistry.
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INTRODUCTION |
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Carbon tetrachloride (CCl4) is a well-investigated chemical, and several microarray studies have been published describing gene expression changes caused by acute CCl4 toxicity (Bulera et al., 2001; Harries et al., 2001
). These gene expression profiles have catalogued the molecular responses to acute CCl4 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 CCl4. Furthermore, we examined possible recovery of gene expression changes after removal of CCl4 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 CCl4 treatment, and most of these changes can be reversed upon treatment cessation. However, some changes were persistent. In particular, CCl4-induced up-regulation of genes involved in liver fibrogenesis, which correlated well with the prolonged fibrotic changes in the liver, remained after CCl4 withdrawal.
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MATERIALS AND METHODS |
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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 µm 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 µm 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-ß1(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® 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 µg 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 2x standard saline citrate (SSC) containing 1% sodium dodecyl sulfate for 30 min followed by 2 washes with 0.1x 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 (real-time 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 genes 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 Duncans multiple range tests. Differences were considered significant when p < 0.05.
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RESULTS |
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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 CCl4 treatment for 4 weeks, and most of these gene expressions remained at the high levels after CCl4 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 CCl4 for 4 weeks. Some of these genes still had a higher level of expression 4 weeks after the cessation of the CCl4 treatment. Certain stress/DNA damage genes also showed increased expression after CCl4 treatment for 4 weeks and decreased to the level of controls after CCl4 withdrawal for 4 weeks, with the exception of GADD153. The CYP2E1 gene showed a two-times decreased expression after CCl4 treatment for 4 weeks and regained its normal expression level after cessation of CCl4 treatment for 4 weeks.
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DISCUSSION |
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Hepatotoxicity of CCl4 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 CCl4 (Pietrangelo, 1996
). Acute administration of CCl4 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 CCl4-treated animals were the expression of genes involved in stress, DNA damage, proliferation, and metabolic enzymes. Similar to the acute administration, treatment with CCl4 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 CCl4 insults. Most of the affected gene expressions in these two categories were returned to untreated control levels after CCl4 withdrawal for 4 weeks. The exceptions were that GADD153, c-myc, and Weel/p87 remained at high levels of expression 4 weeks after cessation of CCl4 treatment, indicating that the regulatory mechanisms that increased by CCl4 treatment remained in an active state. These genes might serve as markers to monitor the recover of liver injury.
CCl4 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 CCl4 resulted in down-regulation of CYP2E1 (McGregor and Lang, 1996
; Pietrangelo, 1996
; Waring 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 CCl4 for 4 weeks and did not showed any recovery after cessation of CCl4 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 CCl4 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 CCl4 in the liver. Interestingly, it has been shown that the activities of these enzymes were inhibited by a high dose (1000 µl/kg) of acute CCl4 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 CCl4-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 CCl4, 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 CCl4 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 CCl4 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 CCl4, 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 CCl4. 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 CCl4 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 cessation of the treatment with CCl4, which would be highly responsible for the persistent fibrosis in the liver.
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ACKNOWLEDGMENTS |
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NOTES |
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1 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: yjkang01{at}louisville.edu
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