A temporal study on the histopathological, biochemical and molecular responses of CCl4-induced hepatotoxicity in Cyp2e1-null mice

Toxicology 228 (2006) 310–322

A temporal study on the histopathological, biochemical and molecular responses of CCl4-induced hepatotoxicity in Cyp2e1-null mice Sreedevi Avasarala a , Lei Yang a , Yan Sun a , Alice Wan-Chi Leung b , Wood-Yee Chan c , Wing-Tai Cheung a , Susanna Sau-Tuen Lee a,b,∗ a

b

Department of Biochemistry, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China Environmental Science Programme, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China c Department of Anatomy, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China Received 15 September 2006; accepted 28 September 2006 Available online 6 October 2006

Abstract Previous study using Cyp2e1-null mice showed that Cyp2e1 is required in CCl4 -induced liver injury at 24 h, what remains unclear are the temporal changes in liver damage and the spectrum of genes involved in this process. We investigated the time-dependent liver changes that occurred at morphological, histopathological, biochemical and molecular levels in both Cyp2e1+/+ and Cyp2e1−/− mice after treating with either corn oil or CCl4 (1 ml/kg) for 2, 6, 12, 24 and 48 h. A pale orange colored liver, indicative of fatty infiltration, was observed in Cyp2e1+/+ mice treated with CCl4 for 24 and 48 h, while the Cyp2e1+/+ mice treated with corn oil and Cyp2e1−/− mice treated with either corn oil or CCl4 showed normal reddish brown colored liver. Ballooned hepatocytes with multiple vacuoles in their cytoplasm were observed in the livers of Cyp2e1+/+ mice 24 and 48 h after treating with CCl4 . The levels of serum alanine aminotransferase and aspartate aminotransferase, markers for liver injury, were significantly higher at 12 h, peaked at 24 h and gradually decreased at 48 h after CCl4 intoxication. In contrast, this kind of damage was not apparent in the Cyp2e1−/− mice treated with CCl4 . Altered expressions of genes related to liver cirrhosis, apoptosis, oxidative stress, xenobiotic detoxification, lipid metabolism, chemsensory signaling or tumorigenesis, structural organization, regeneration and inflammatory response were identified, and the time-dependent changes in expression of these genes were varied. Overall, the present study provides insights into the mechanism of CCl4 -induced hepatotoxicity in animal models. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Carbon tetrachloride; Cyp2e1-null mice; Fluorescent differential display; Gene expression profiling; Oxidative stress

1. Introduction Carbon tetrachloride (CCl4 ) is the classic model for free radical-induced liver injury. Through the investigation of acute CCl4 -induced liver damage in ani-

∗

Corresponding author. Tel.: +852 2609 6333; fax: +852 2603 7818. E-mail address: [email protected] (S.S.-T. Lee).

mal models in the past 50 years, it is now generally accepted that CCl4 toxicity results from bioactivation of the CCl4 molecule to the trichloromethyl free radical by cytochrome P450 isozymes (P450s). The trichloromethyl radical then reacts with oxygen to form the highly toxic reactive trichloromethyl peroxy radical. The free radicals subsequently attack on the polyunsaturated fatty acids of membrane lipids to propagate a chain reaction resulting in breakdown of membrane structure,

0300-483X/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2006.09.019

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disrupting cell energy processes and protein synthesis (Recknagel et al., 1989; Plaa, 2000; Weber et al., 2003). Among the numerous cytochrome P450s present in liver, cytochrome P450 2E1 (Cyp2e1) has been demonstrated to play a major role in CCl4 toxicity based on a previous study with Cyp2e1-null mice in our laboratory (Wong et al., 1998). These mice that lack Cyp2e1 protein expression were resistant to CCl4 -induced hepatotoxicity 24 h after CCl4 intoxication. Although this study clearly demonstrates that Cyp2e1 plays an important role in CCl4 -induced liver damage, what remains unclear are the temporal liver changes that occur at morphological, histopathological and biochemical levels. At molecular levels, several studies have been reported describing gene expression changes caused by acute or chronic CCl4 treatment (Bulera et al., 2001; Harries et al., 2001; Jiang et al., 2004). Acute administration of CCl4 to rats causes significant changes in gene expression profiles (Waring et al., 2001; Fountoulakis et al., 2002). It is evident from these studies that some genes appear transiently affected, while others show a persistent alteration, depending upon the dose and time of exposure. CCl4 -induced liver injury occurs mainly at two levels (Bruccoleri et al., 1997). The primary CCl4 induced liver injury has been linked to genes involved in different pathological processes such as oxidative stress, necrosis, apoptosis and fibrosis. In contrast, secondary CCl4 -induced liver injury is mainly mediated by release of cytokines and interleukins from the Kupffer and stellate cells. However, there is no comprehensive report regarding the temporal changes of these genes in CCl4 induced hepatotoxicity in animal models. To extend our previous study on using Cyp2e1-null mouse model to understand the mechanism of CCl4 induced hepatotoxicity (Wong et al., 1998), we analyzed the time-dependent liver changes that occurred at morphological, histopathological, biochemical and molecular levels in both Cyp2e1+/+ and Cyp2e1−/− mice (Lee et al., 1996) after treating with either corn oil or CCl4 (1 ml/kg) for 2, 6, 12, 24 and 48 h. 2. Materials and methods 2.1. Animals Cyp2e1-null mice (Cyp2e1−/− ) and their wild-type (Cyp2e1+/+ ) counterparts are inbred strains on SV/129/ter genetic background (Lee et al., 1996). A pair of parental stocks of Cyp2e1+/+ and Cyp2e1−/− mice was shipped from the National Cancer Institute (National Institutes of Health, Bethesda, MD, USA). They were then bred and reared at the Chinese University of Hong Kong animal rooms on a 12h light:12-h dark cycle (light, 06:00–18:00) period. Animals

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were fed with laboratory mouse diet No. 5015 (PMI Nutrition Inc., St. Louis, MO, USA) and chlorinated tap water ad libitum. 2.2. CCl4 treatment Carbon tetrachloride (99.8% purity, Riedell-de Haen, Germany) and corn oil (Lion and Globe Company, Hong Kong) were used for treating the animals. The detail procedures for treatment of animals with CCl4 were given in our earlier work (Wong et al., 1998). Briefly, 3 months old male Cyp2e1+/+ and Cyp2e1−/− mice (20–30 g) were acclimatized for at least 1 week prior to the experiments. The Cyp2e1+/+ and Cyp2e1−/− mice (n = 5–12 per treatment) were i.p. injected with 1 ml/kg body weight (=1.59 g/kg) CCl4 (10% solution in corn oil). Controls for each group were injected with corn oil vehicle only. Mice were sacrificed by decapitation and blood was collected. Livers were excised, weighed, immediately frozen and stored in liquid nitrogen until further used for RNA preparation. 2.3. Determination of serum aminotransferase activities Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, markers for hepatotoxicity, were determined enzymatically by the rate of NADH oxidation with reagent kits obtained from Stanbio Laboratory (Boerne, TX, USA). 2.4. Liver histology A small liver section was excised from the median lobe of each mouse for liver histopathology analysis. Three liver samples were collected for each treatment group. The tissues were fixed in 10% phosphate buffered formalin for 48 h and then transferred to 70% ethyl alcohol, processed and embedded in paraffin. Liver sections (5 ␮m thick) were stained with hematoxylin-eosin (H&E) for histological examination under a light microscope (Axiophot 2, Zeiss, Germany). 2.5. mRNA differential display The protocols for mRNA differential display were discussed elaborately in our earlier publication (Lee et al., 2002). In brief, the fluorescent differential display (FDD) procedures involved extraction of total liver RNA by TRIzol® (Invitrogen, Carlsbad, CA, USA) reagent according to the manufacturer’s instructions and the extracted RNA was treated with RNase-free DNase I (Amersham Biosciences, Piscataway, NJ, USA) to remove the possible genomic DNA contamination. RNA from at least two different mice was isolated for each treatment group. The HIEROGLYPH mRNA profile and FluoroDD kits (Beckman Coulter, Inc., Fullerton, CA, USA) were used to synthesize subpopulation of first-strand cDNAs from the total liver RNA isolated, and those cDNAs were subsequently amplified by PCR. In the first

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step of reverse transcription, two different RNA samples each from Cyp2e1+/+ /corn oil, Cyp2e1+/+ /CCl4 , Cyp2e1−/− /corn oil, or Cyp2e1−/− /CCl4 were served as templates for the first-strand cDNA synthesis in the presence of 0.2 ␮M T7 oligo(dT) two-based 3 anchored primer (AP), 1× first-strand buffer, 25 ␮M dNTP mix (1:1:1:1), 10 mM DTT and 2 units of SuperScriptTM II Rnase H− reverse transcriptase (Invitrogen, Carlsbad, CA, USA). The cDNAs produced in the reverse transcription reactions were used in the PCR reactions in the presence of 4 ␮l of first-stranded reverse transcribed cDNA mixture, 1× PCR buffer, 50 ␮M dNTP mix (1:1:1:1), 0.35 ␮M 5 -arbitrary primer (ARP), 0.35 ␮M 3 -AP (fluoresceinlabeled) and 0.05 units of Taq DNA polymerase (Genesys Ltd., Surrey, UK). The conditions for PCR included 95 ◦ C for 2 min, 4 cycles at 92 ◦ C for 15 s, 50 ◦ C for 30 s, 72 ◦ C for 2 min and 30 cycles at 92 ◦ C for 15 s, 60 ◦ C for 30 s and 72 ◦ C for 2 min. Six different combinations of APs and ARPs were used in performing the FDD-PCR reactions. For gel SA, AP7 (5 -ACGACTCACTATAGGGCTTTTTTTTTTTTCG-3 ) and ARP10 (5 -ACAATTTCACACAGGAGATCTCAGAC-3 ); gel SB, AP12 (5 -ACGACTCACTATAGGGCTTTTTTTTTTTTCT-3 ) and ARP6 (5 -ACAATTTCACACAGGATACAACGAGG-3 ); gel SC, AP8 (5 -ACGACTCACTATAGGGCTTTTTTTTTTTTAA-3 ) and ARP2 (5 -ACAATTTCACACAGGAGCTAGCATGG-3 ); gel SF, AP5 (5 -ACGACTCACTATAGGGCTTTTTTTTTTTTCA-3 ) and ARP1 (5 -ACAATTTCACACAGGACGACTCCAAG-3 ); gel A, AP5 (5 -ACGACTCACTATAGGGCTTTTTTTTTTTTCA-3 ) and ARP2 (5 -ACAATTTCACACAGGAGCTAGCATGG-3 ) and gel B, AP5 (5 -ACGACTCACTATAGGGCTTTTTTTTTTTTCA-3 ) and ARP4 (5 -ACAATTTCACACAGGAGCTAGCAGAC-3 ) were used. The TMR-labeled fluorescent PCR products and TMR molecular weight marker were electrophoresed on 5.6% denatured polyacrylamide gels at 3000 V, 100 W and 50 ◦ C for approximately 4 h using a GenomyxLRTM DNA sequencer electrophoresis system (Beckman Coulter, Inc., Fullerton, CA, USA). After electrophoresis, the FDD gels were scanned in a GenomyxSC fluorescent imaging scanner (Beckman Coulter, Inc., Fullerton, CA, USA) for analysis of the cDNA band patterns on the differential display gels. The fluorescent differential gene expression patterns were compared across treatment and mouse groups to confirm a consistent pattern of changes. Since we were interested in the Cyp2e1-associated CCl4 -induced liver damage, the cDNA fragments that showed differential expression patterns in Cyp2e1+/+ but not in Cyp2e1−/− mice after 24 h CCl4 treatment were carefully excised from the FDD gels, eluted in Tris–EDTA (TE) buffer and reamplified by PCR (Lee et al., 2002). The amplified fragments were then cloned directly into a pCR® II-TOPO TA cloning vector (Invitrogen, Carlsbad, CA, USA). The clones with the correct insert size were chosen for Taq DyeDeoxy terminator cycle sequencing with a CEQTM 2000 DNA analysis system (Beckman Coulter, Inc., Fullerton, CA, USA). The sequences were then compared with the known sequences by using DNA databases at the National Center for Biotechnology Information and BLAST server.

2.6. Northern blot analysis The subcloned FDD cDNA fragments were used as probes to confirm their differential expressions by Northern blot analysis. RNA from different batch of mice was used in the Northern blot analysis. For the temporal gene expression study, RNA from 2, 6, 12, 24 and 48 h after CCl4 exposure was used. Total RNA was extracted from 2–3 corn oil- or CCl4 -treated liver samples from both the Cyp2e1+/+ and Cyp2e1−/− mice. Northern blot analysis was performed as reported earlier (Lee et al., 2002). Briefly, electrophoretic separation of RNA was carried out on a 1% formaldehyde agarose gel and then transferred to a positively charged nylon membrane (Pall Corporation, East Hills, NY). The FDD cDNA fragments were then used for preparing the probes by PCR dioxygenin (DIG)-labeling (Roche Diagnostics, Penzberg, Germany). Hybridization was performed at 42 ◦ C overnight. The blots were then washed at room temperature twice in 2× SSC, 0.1% SDS for 15 min and at 68 ◦ C in 0.5× SSC, 0.1% SDS for 15 min. After washing, the blots were blocked in a blocking buffer (Roche Diagnostics, Penzberg, Germany) and then incubated in an anti-DIG antibody alkaline phosphatase conjugate (1:10,000) solution for 1 h at room temperature. The blots were then incubated in a detection buffer containing nitroblue tetrazolium and 5-bromo4-chloro-3-indolyl-phosphate (Roche Diagnostics, Penzberg, Germany) for several to 24 h to develop the signals on the blots. Color development was terminated with autoclaved distilled water and the blots were scanned with an Epson Expression 1600 Pro Scanner at a resolution of 300 dpi, and the digitalized images were captured and processed with the Photoshop 6.0 software. 2.7. Statistical analysis Data are expressed as mean ± S.D. Differences in treatment means for the two genotypes of mice were analyzed by using a SigmaStat Advisory Statistical Software (SigmaStat Version 3.1; SPSS Inc., Chicago, IL, USA). Each set of data was analyzed with one way ANOVA if the data set passed normality and equal variance tests. Otherwise, Kruskal–Wallis one way ANOVA on ranks was used to compare the differences in mean values within each time-point and also between time-points. Dunn’s and Tukey’s tests were performed where appropriate to assess the differences in treatment means. Statistical significance was determined at a level of P < 0.05.

3. Results 3.1. Morphological observations At morphological level, the extent of CCl4 -induced liver injury was analyzed by observing the mortality, liver weight and liver colors of the Cyp2e1+/+ and Cyp2e1−/− mice treated with either corn oil vehicle or CCl4 (1 ml/kg). In this experiment, 100% survival was

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CCl4 for 24 and 48 h, while the Cyp2e1+/+ mice treated with corn oil and Cyp2e1−/− mice treated with either corn oil or CCl4 showed normal reddish brown color (Fig. 1). The pale orange color observed in the livers of Cyp2e1+/+ animals was an apparent indication of fatty infiltration resulting from the exposure to CCl4 . Thus, these observations strongly support that at morphological level fatty infiltration has started at 24 h and continued till 48 h in the Cyp2e1+/+ but not in mice lacking the Cyp2e1 protein following CCl4 intoxication. 3.2. Liver histopathological observations

Fig. 1. Liver morphology of Cyp2e1+/+ and Cyp2e1−/− mice treated with either corn oil vehicle or 1 ml/kg CCl4 for 2, 6, 12, 24 and 48 h. Data shown are representative of three separate experiments (n = 9 per treatment) with similar results.

noted as both the Cyp2e1+/+ and Cyp2e1−/− mice administered with either corn oil or 1 ml/kg CCl4 for 2, 6, 12, 24 and 48 h tolerated the treatments (data not shown). This data was consistent with earlier studies in Cyp2e1-null mice (Wong et al., 1998) and male Sprague–Dawley rats (Rao et al., 1997) that no mortality was observed in these animals receiving a single dose of CCl4 up to 1 ml/kg bw at 24 h and 14 days, respectively. There were no obvious differences in the liver weight (as percentage of body weight) between the Cyp2e1+/+ and Cyp2e1−/− mice treated with either corn oil or CCl4 at all time-points (data not shown). The liver color appeared normal reddish brown in both Cyp2e1+/+ and Cyp2e1−/− mice treated with either corn oil or CCl4 for 2, 6 and 12 h (Fig. 1). A visible and distinct color change (orange color) was observed in the livers of Cyp2e1+/+ mice treated with

CCl4 administration to the Cyp2e1+/+ mice for 2, 6 and 12 h caused no obvious histological abnormalities in the livers of these mice (Fig. 2A and B). However, drastic histological damage was observed in the livers of Cyp2e1+/+ mice 24 and 48 h following CCl4 treatment. In these livers, the hepatic lobules became less eosinophilic especially in the central vein region. In the central vein region many liver cells were ballooned with multiple vacuolation in their cytoplasm. The arrangement of the anastomosing plates of hepatocytes was disrupted. The hepatic sinusoids appeared to be collapsing and diminished in size. Degenerating cells with pyknotic nuclei were found in the region surrounding the central vein. The nuclei were normal in the portal vein region. Ballooning of hepatocytes and degenerative nuclei were not observed in the portal vein region. Thus, the injury appeared to be confined to the central vein region. In the Cyp2e1−/− mice, CCl4 treatment for 24 and 48 h did not result in any noticeable histological changes. Similar observations were found in Cyp2e1+/+ and Cyp2e1−/− mice treated with corn oil vehicle only at all time-points. 3.3. Serum aminotransferase activities The extent of CCl4 -induced liver injury was also examined by serum ALT and AST levels. In the Cyp2e1+/+ mice, the fold increases in serum ALT levels for 2, 6, 12, 24 and 48 h following CCl4 treatment were 1.00, 1.34, 17.3, 52.4 and 7.11, respectively, while the fold increases in serum AST levels were 1.04, 1.32, 6.08, 268 and 15.0, respectively, as compared to their corresponding corn oil-treated controls. At 2 and 6 h, the serum ALT (Fig. 3A) and AST (Fig. 3B) levels in the Cyp2e1+/+ mice treated with CCl4 were not statistically significant as compared to their corresponding corn oil controls. Significantly higher serum ALT and AST levels were noted in the Cyp2e1+/+ mice starting from 12 to 48 h with a maximum peak at 24 h following CCl4 poisoning.

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Fig. 2. Representative H&E-stained histological sections of livers from Cyp2e1+/+ and Cyp2e1−/− mice 2, 6, 12, 24 and 48 h following treatment with 1 ml/kg CCl4 or corn oil vehicle (n = 3 per treatment). (A) Low power magnification 10×. (B) High power magnification 40×. C, central vein; scale bars, 50 ␮m.

On the contrary, at all time-points the CCl4 treatment in Cyp2e1−/− mice did not result in any significant increase in the serum ALT and AST levels as compared to their corresponding corn oil-treated controls. 3.4. Gene expression analysis To identify the genes responsible for Cyp2e1associated, maximum CCl4 -induced liver injury, mRNA differential display was performed using the total RNA isolated from the livers of both corn oil and CCl4 -treated Cyp2e1+/+ and Cyp2e1−/− mice at 24 h. Six fluorescent differential display (FDD) gels (SA, SB, SC, SF, A and B) were performed and differentially expressed fragments (as indicated by arrows) were excised, subcloned and sequenced (Fig. 4). Interestingly, the ∼675 bp cDNA fragment in FDD gel SF consisted of two different comigrating gene fragments (Tubb 2 and Chka) as revealed by subcloning and sequencing. The mRNA differential display revealed significant changes in liver gene expressions in the Cyp2e1+/+ mice treated with CCl4 . From the 10 cDNA fragments identified from the six FDD gels, six of them were down-regulated [interleukin-1 receptor

accessory protein (IL-1RAcP), phenylalanine hydroxylase (Pah), cytochrome P4501A2 (Cyp1a2), BCL-2 and adenovirus 19 kDa-interacting protein (Bnip3), carbonic anhydrase III (Car3) and major urinary protein (MUP)], while the other four gene fragments [tubulin (Tubb 2), choline kinase alpha (Chka), adipocyte differentiation related protein (ADRP) and gamma actin (Actg)] were up-regulated in the Cyp2e1+/+ mice treated with 1 ml/kg CCl4 for 24 h. In contrast, the expression of all these cDNA fragments was not significantly affected in the Cyp2e1−/− mice receiving the same treatment, suggesting that these genes are involved in the Cyp2e1associated CCl4 -induced liver injury. The differential expression patterns of the cDNA fragments identified from the FDD gels were further confirmed by Northern blot analysis using their corresponding cDNA fragments as probes. In agreement to the differential expression patterns in the FDD gels (Fig. 4), the mRNA expression levels of IL-1RAcP, Pah, Cyp1a2, Bnip3 and Car3 (Fig. 5A) and MUP (Fig. 5B) were down-regulated in the Cyp2e1+/+ mice treated with CCl4, while such down regulations were not observed in the corresponding Cyp2e1−/− group receiving the

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Fig. 3. Serum alanine aminotransferase (A) and aspartate aminotransferase (B) of Cyp2e1+/+ and Cyp2e1−/− mice after 2, 6, 12, 24 and 48 h exposure to 1 ml/kg CCl4 or corn oil vehicle (CTL). Data are expressed as mean ± SD (n = 5–12). * Significant difference in the CCl4 group as compared to the corn oil control. NS, indicates no significant difference.

same treatment. Similarly, up-regulation of Tubb 2, Chka, ADRP and Actg mRNA levels was found in the Cyp2e1+/+ mice treated with CCl4 (Fig. 5B), which were consistent with the differential gene expression patterns in the FDD gels (Fig. 4). The temporal expression patterns of the 10 genes identified in both Cyp2e1+/+ and Cyp2e1−/− mice treated with CCl4 were varied. Down-regulation of IL1RAcP, Pah, Cyp1a2, Bnip3, Car3 and MUP distinctively started at 12 h, peaked at 24 h and continued at 48 h in the Cyp2e1+/+ mice treated with CCl4, while similar constitutive expressions of IL-1RAcP, Pah, Cyp1a2,

Bnip3 and MUP were observed in Cyp2e1−/− mice treated with CCl4 at all time points (Fig. 6A and B). Interestingly, down-regulation of Car3 was also observed in the Cyp2e1−/− mice 12 and 48 h following CCl4 treatment. The time-dependent up-regulation of Tubb 2, Chka and Actg commenced as early as 2 h and peaked at 2, 12 and 24 h for the Actg, Chka and Tubb 2, respectively, in the CCl4 -treated Cyp2e1+/+ mice (Fig. 6B). There was a delay in the up-regulation of ADRP at 6 h with a maximum peak at 24 h in the Cyp2e1+/+ mice following CCl4 treatment. Constitutive expressions of Tubb 2, Chka, ADRP and Actg were observed in Cyp2e1−/−

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Fig. 4. mRNA differential display of liver RNA from Cyp2e1+/+ and Cyp2e1−/− mice treated with either corn oil vehicle (CTL) or 1 ml/kg CCl4 for 24 h. Total RNA was first reverse transcribed with anchored primers (APs). Subsequent PCR reactions were performed in duplicate for each mouse in the presence of 3 -TMR-labeled APs and 5 -arbitrary primers (APRs). The APs and ARPs used in gels SA, SB, SC, SF, A and B were as described in Section 2. The TMR-labeled fluorescent PCR products were separated on 5.6% denaturing polyacrylamide gels. Each treatment group includes two mice with duplicate PCR (1 and 2) reactions. Arrows indicate cDNA fragments that were excised, reamplified, subcloned and sequenced. IL-1RAcP, interleukin-1 receptor accessory protein; Pah, phenylalanine hydroxylase; Cyp1a2, cytochrome P4501A2; Bnip3, BCL-2 and adenovirus 19 kDa-interacting protein; Car3, carbonic anhydrase III; Tubb 2, tubulin; Chka, choline kinase alpha; MUP, major urinary protein; ADRP, adipocyte differentiation related protein; Actg, gamma actin. M, TMR-labeled molecular weight DNA marker.

mice treated with CCl4 . However, the Cyp2e1-/- mice also showed mild increased expressions of Tubb 2, Chka and Actg at 6 and 12 h following CCl4 treatment. Here we used the ethidium bromide stained formaldehyde gels as loading controls because it has been reported

that CCl4 affects the mRNA expression levels of house keeping genes including glyceraldehyde-3-phosphate dehydrogenase (Itoh et al., 1992), ␤-actin and albumin (Armendariz-Borunda et al., 1990; Goldsworthy et al., 1993; Nakamura et al., 1994).

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Fig. 5. Confirmation of differential expression patterns of identified genes by Northern blot analysis. Thirty micrograms of total liver RNA from Cyp2e1+/+ and Cyp2e1−/− mice treated with either corn oil (CTL) or 1 ml/kg CCl4 for 24 h (n = 3 per treatment) were separated on 1% formaldehydeagarose gels, transferred to nylon membranes and hybridized with their corresponding PCR digoxigenin-labeled cDNA fragments. The signal was detected by a colorimeteric method with reagents nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate. The intensity of 28S and 18S in the ethidium bromide-stained agarose gel indicates that approximate equal amounts of total RNA were loaded, and each lane represents RNA sample from an individual mouse. Data shown is a representative of two separate experiments with similar results. (A) IL-1RAcP, interleukin-1 receptor accessory protein; Pah, phenylalanine hydroxylase; Cyp1a2, cytochrome P4501A2; Bnip3, BCL-2 and adenovirus 19 kDa-interacting protein; Car3, carbonic anhydrase III. (B) Tubb 2, tubulin; Chka, choline kinase alpha; MUP, major urinary protein; ADRP, adipocyte differentiation related protein; Actg, gamma actin. M, RNA molecular weight marker I, digoxygenin-labeled (0.39–6.9 kb).

4. Discussion After 24 and 48 h, a pale orange colored liver was observed in the Cyp2e1+/+ mice treated with a single dose of 1 ml/kg CCl4 . Similar kind of color change was observed in our previous finding in Cyp2e1+/+ mice treated with CCl4 for 24 h (Wong et al., 1998). This pale color changes observed in the Cyp2e1+/+ mice treated with CCl4 appeared due to fatty infiltration resulting from exposure to CCl4 (Paquet and Kamphausen, 1975; Cunnane, 1987). It has been reported that fatty accumulation in the liver following CCl4 poisoning is the result of imbalance between lipid synthesis and degradations, and failure of triglycerides to move as very low-density lipoproteins from liver to the circulation (Boll et al., 2001a,b). In contrast, the liver color was reddish brown in Cyp2e1−/− mice treated with CCl4 , supporting the earlier finding (Raucy et al., 1993) that Cyp2e1 is required in bioactivation of CCl4 to products that lead to fatty

accumulation in livers of animals in a time-dependent manner. In line with the time-dependent accumulation of lipids in the livers of Cyp2e1+/+ mice treated with 1 ml/kg CCl4 , the time course study indicated that there was no obvious liver injury observed till 12 h and severe centrilobular necrosis was limited in the region surrounding the central vein in the Cyp2e1+/+ mice 24 and 48 h following the CCl4 intoxication. Similar observations were reported in time course studies conducted in rats. In Wistar rats, hydropic swelling and focal necrosis were observed in the centriblobular region as early as 3 h after 2 ml/kg CCl4 treatment, peaked at 24 h, continued till 48 h and thereafter gradually reduced (Wang et al., 1997). In Sprague–Dawley rats, centrilobular injury was evident as early as 6 h with maximum injury at 36 following 1 ml/kg CCl4 intoxication (Rao et al., 1997). It has been reported that the highest concentration of Cyp2e1 occurs in the central vein region of liver (Ingelman-

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Sundberg et al., 1988; Tsutsumi et al., 1989; Buhler et al., 1991) and CCl4 is bioactivated by Cyp2e1 to reactive free-radicals which then trigger the cascade of free-radical-induced cetnrilobular liver damage. Similar observations were noted in the cells surrounding the central vein region during the liver damage caused by ethanol, other halogenated hydrocarbons and other Cyp2e1-activated substrates (Lieber, 1997; Rao et al., 1997). On the other hand, the Cyp2e1−/− mice were resistant to the CCl4 -induced liver injury as the Cyp2e1mediated CCl4 bioactivation pathway was completely blocked in these mice. Taken together, these data suggest that Cyp2e1 is required in the biotransformation of CCl4 to products that lead to a time-dependent onset of centrilobular liver injury with the maximum damage at 24–36 h depending on the dosage and animal species. Biochemical alterations are likely to occur well before significant histological damage is evident (Weber et al., 2003). In our study, the injury at biochemical level was assessed by measuring the activities of two liver specific marker enzymes namely serum ALT and AST. The serum ALT and AST activities are found within the hepatocytes. An elevation of these enzyme levels in blood may represent conditions that alter the permeability of cell membrane to such a degree that ALT and AST leak into the blood serum, and the increase is proportional to the number of affected hepatocytes (Hall, 1992). Similar to an earlier study in rat (Wang et al., 1997), the serum ALT and AST levels were significantly increased at 12, 24 and 48 h in the CCl4 -treated Cyp2e1+/+ mice as compared to the Cyp2e1−/− mice receiving the same treatment. The activities of these enzymes were gradually increased from the initial time-points to 12 h, peaked at 24 h and then gradually reduced at 48 h indicating the extent of liver damage at different time-points. Whereas, the histopathological studies showed that the injury started at 24 h and continued till 48 h in the Cyp2e1+/+ mice treated with CCl4 . These results clearly demonstrate that injury at biochemical level starts well before the first appearance of histopathological symptoms. At molecular level, our mRNA differential display study revealed that 24 h following 1 ml/kg CCl4 treat-

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ment resulted in altered expression of many genes involved in several important cellular processes such as liver cirrhosis [Pah (Ishii et al., 2001)]; apoptosis [Bnip3 (Guo et al., 2001)]; oxidative stress [Car3 (Parkkila et al., 1999; Yamamoto et al., 2006)]; xenobiotic detoxification [Cyp1a2 (Smith et al., 2003)]; lipid metabolism [ADRP (Ye and Serrero, 1998)]; chemosensory signaling or tumorigenesis [MUP II (Dragani et al., 1989; Armstrong et al., 2005)]; structural organization [Tubb 2 (Toh et al., 1977; Yamamoto et al., 2006), Actg (Lee et al., 1998)]; regeneration [Chka (Houweling et al., 1991)] and inflammatory response [IL-1RAcP (Jensen et al., 2000)]. Among the 10 genes identified in the present study, six of them (IL-1RAcP, Pah, Cyp1a2, Bnip3, Car3 and MUP) were down-regulated upon treatment with a single dose of CCl4 . The onset of down-regulation was quickly, peaked at 12–24 h and lasted for at least 48 h. This temporal pattern of gene down-regulation is in parallel with the time-dependent changes of serum AST and ALT levels, suggesting that those down-regulated genes are directly linked to CCl4 -induced hepatic toxicity. It is of interest to note that Tubb 2, Chka, ADRP and Actg were significantly up-regulated in wild-type mice within 2–6 h of CCl4 intoxication and their expressions were subsequently returned to the control level during the recovery phase at 48 h. However, a mild increase of Tubb 2, Chka and Actg expressions was also observed but at later time points at 6–12 h and then quickly returned to the control levels at 24 h following CCl4 intoxication in the Cyp2e1-deficient mice, suggesting that these gene changes are not due to the sole effect resulting from the Cyp2e1-mediated bioactivation of CCl4 . It has been demonstrated that Cyp2e1 is the major cytochrome P450s to execute biotransformation of CCl4 (Raucy et al., 1993), but other cytochrome P450s including CYP1B1 and CYP2B2 (Gruebele et al., 1996) and CYP3A (Zangar et al., 2000) are also capable of bioactivating CCl4. Taken together, these results suggest that the extent of acute CCl4 -induced liver injury depends on the magnitude of CCl4 bioactivation as well as the spectrum and temporal expression patterns of genes being affected.

Fig. 6. Temporal expression patterns of identified genes by Northern blot analysis. Thirty micrograms of total liver RNA from Cyp2e1+/+ and Cyp2e1−/− mice treated with either corn oil (CTL) or 1 ml/kg CCl4 for 2, 6 12, 24 and 48 h (n = 2 per treatment) were separated on 1% formaldehyde–agarose gels, transferred to nylon membranes and hybridized with their corresponding PCR digoxigenin-labeled cDNA fragments. The signal was detected by a colorimeteric method with reagents nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate. The intensity of 28S and 18S in the ethidium bromide-stained agarose gel indicates that approximate equal amounts of total RNA were loaded, and each lane represents RNA sample from an individual mouse. Data shown are representative of two separate experiments with similar results. (A) IL-1RAcP, interleukin-1 receptor accessory protein; Pah, phenylalanine hydroxylase; Cyp1a2, cytochrome P4501A2; Bnip3, BCL-2 and adenovirus 19 kDa-interacting protein; Car3, carbonic anhydrase III. (B) Tubb 2, tubulin; Chka, choline kinase alpha; MUP, major urinary protein; ADRP, adipocyte differentiation related protein; Actg, gamma actin. M, RNA molecular weight marker I, digoxygenin-labeled (0.39–6.9 kb).

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Alteration in gene expression profiles caused by acute treatment of CCl4 has recently been studied using microarrays (Bulera et al., 2001; Harries et al., 2001; Jiang et al., 2004). In comparison of the present mRNA differential display with those microarray studies, however, it is of interest to note that no identical gene was found among all those microarray studies as well as the present mRNA differential display, though isolated genes do share similar functions. For example, detoxification enzyme cytochrome P450 was up-regulated after CCl4 treatment, but different P450 isoforms were identified in different studies. In general, the expression of proteins and enzymes involving in xenobiotic metabolism, apoptosis, lipid metabolism, oxidative stress and cytoskeleton was altered after CCl4 treatment. Different genes were identified among various studies and this is partly due to the fact that only genes differentially altered in the wild-type but not in Cyp2e1-null mice upon 24 h CCl4 exposure were identified in our study. Alternatively, different CCl4 treatment dosages and regimes, different animal species as well as RNA samples collected at different time points among different studies may also contribute to the different genes that were identified. Among the genes identified in the present study, it is noted that expression of MUP is repressed by activation of PPAR␣ with Wy-14,643 (Motojima et al., 1997), while ADRP is stimulated by indomethacin (Ye and Serrero, 1998), an activator of PPAR␣ and γ (Lehmann et al., 1997). Recently, PPAR␣ was found to mediate the induction of various xenobiotic metabolizing enzymes in mouse intestine and liver (Motojima and Hirai, 2006). Though the hepatic toxicity of CCl4 has been suggested to be mediated by Cyp2e1 via free radical formation, the down-stream mechanistic nature of hepatic responses is still largely unknown. Hence, it would be of interest to test whether the activation of PPAR␣ would contribute partly to the hepatic response upon CCl4 challenge using the PPAR␣-deficient mouse model (Lee et al., 1995). In conclusion, the present findings based on the morphological, histopathological, biochemical and molecular data indicated that Cyp2e1 is required in mediating the biotransformation of CCl4 to reactive products that trigger the cascade of liver injury in a timedependent manner. CCl4 -induced liver injury was evident at 12 h and maximum injury was noted at 24 h which followed by a gradual recovery at 48 h. The timedependent liver damages upon CCl4 intoxication and possibly liver regeneration are related to the differential temporal expression of genes related to liver cirrhosis, apoptosis, oxidative stress, xenobiotic detoxification, lipid metabolism, chemsensory signaling or tumorigen-

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