The level of TGF-β1 was assessed by using four independent methods: analysis of TGF-β1 in plasma (Fig. 1A), mRNA (Fig. 1B) and protein (Fig. 1C) in the liver and immunohistochemical staining in liver sections (Fig. 1D). The results indicate that serum levels of TGF-β1 in pPK9a-transferred mice fed with ZnSO₄ peaked at 48 h and were higher than the four control groups (Fig. 1A). In the absence of ZnSO₄ and pPK9a the serum TGF-β1 level is much lower than in their presence (Fig. 1A). The serum TGF-β1 values fell between 600 and 900 pg/ml and were 5 to 15 times higher than the controls (Fig. 1A); the peak was followed immediately by a decline at the 72^nd h even when ZnSO₄ was not withdrawn. The level of TGF-β1 declined to its normal level on the 5th day and was no longer inducible on the 7th day (Fig 1E). The results are basically similar to that described by Herweijer et al. who showed time course of gene expression after plasmid DNA gene transfer to the liver: expression of the transferred gene was very high on day1 after portal vein injection of plasmid but diminished quickly by day 2 and declined to low level after day 4 16. Their induction was apparently transient as ours. Moreover, Clouthier et al. demonstrated that upon TGF-β1 gene transfer the mice showed similar pathological morphology in both liver and kidney 17. As observed by them we also find high expression of TGF-β1 in the kidney (data not shown), although in this study we just report our findings on fibrotic events in the liver.

Measurements of the other three analyses were taken at the 48th h and similar results were seen in mRNA and protein expressions in liver tissue (Fig. 1B, C and 1D). Primers specific for porcine TGF-β1 in pPK9a were used for distinguishing transcripts from endogenous mouse TGF-β1 by RT-PCR assay (Fig. 1B). TGF-β1 mRNA was detected in mice with pPK9a-transfer (Fig. 1B lane 1 and 2) but not in those without (Fig. 1B lane 3 and 4). However, in western blot a very trace amount of protein was detected in mice without pPK9a-transfer (Fig. 1C, lane 3 and 4). This could be due to fact that the antibody used in the experiment can recognize TGF-β1 from mice, human and porcine. This trace amount of protein was barely detected in immunohistochemical staining of the liver sections (Fig. 1D II).

The high level of TGF-β1 was accompanied by a strong activation of HSC, as indicated by high expressions of α-SMA and p-Smad 2/3 (Fig. 1C, lane 2). Prominent bands of α-SMA and p-Smad 2/3 were also detected in pPK9a-transferred mice that ingested water in the absence of ZnSO₄ (Fig. 1A, lane 3; 1B, lane 3; 1C, lane 1; and 1D I). It was reported that a very low level of cadmium is consumed from the diet and this metal ion can induce endogenous metallothionein to express to some extent 18. We suspect that the cadmium might cause the endogenous metallothionein induction as shown in Fig. 1C (lane 1). However, the effect of ZnSO₄ on pPK9a became more drastic as shown in Fig. 1D where TGF-β1 was barely detected in D I (without ZnSO₄) as compared to the brilliant staining in D III (with ZnSO₄). The levels of TGF-β1 expression in liver pPK9a-transferred mice treated with ZnSO₄ were upregulated at day 2 and declined at day 3–5 (Fig. 1E). Taken together, the results indicated that TGF-β1 can be markedly induced in pPK9a-tranferred mice treated with ZnSO₄.

Liver sections were sampled from mice with pPK9a transfer at the 24th and 48th h with water containing ZnSO₄ (24, 48 h; Fig. 2A and 2B). The liver samples looked paler and stiffer than that of the control (Fig. 2C and 2D). Using Masson's trichrome staining, we noticed the liver sections from mice with pPK9a transfer and ZnSO₄ induction exhibited a marked perisinusoidal deposition of ECM found mostly in the direct vicinity of large vessels (Fig. 3B, I) and a distinct activation of HSC was also observed as shown by α-SMA expression (Fig. 3A, I). The distribution and intensity of collagen and α-SMA were different from that of the control livers where the two proteins were barely detected (Fig. 3). The sinusoids with an enlarged diameter were observed in Fig. 3B I–III, which was not seen in the control (Fig. 3B, IV). However, a bright red patch is noticeable in TGF-β1-overexpressed liver (Fig. 2B), which appears to be hemorrhagic (Fig. 2B). The "hemorrhage" was observed in most of our livers overexpressing TGF-β1 gene. A similar situation of liver hemorrhage was observed by Clouthier et al. who reported that overexpression of TGF-β1 gene in mice caused not only severe liver fibrosis but also in the extreme case hemorrhage and thrombosis 17. They attributed the extreme situation to the results of overexpression of TGF-β1 and not the triggering cause of TGF-β1 overexpression. We agree with their proposal because we observed a quick increased expression of TGF-β1 followed by a quick decline. Should the TGF-β1 expression was caused by liver damage, we would not have found a prompt decline of TGF-β1 (Fig. 1A and 1E). More research is definitely needed to clarify this controversy.

Forty eight hours after a hydrodynamics-based injection of pPK9a, TGF-β1 was induced by ZnSO₄ and triggered a hepatic injury (Fig. 2 and 3B), resulting in increased alanine transaminase (ALT) levels in the serum of approximately 6 times higher than that of the control groups (Fig. 4A).

The degree of fibrosis was assessed by using three independent methods: the collagen quantitation by measuring hydroxyproline content (Fig. 4B), Col 1A2 mRNA level in liver samples (Fig. 4C) and the histopathological analysis under light microscope (Fig. 3B). The results provide factual evidence that the pPK9a-transferred mice upon induction by ZnSO₄ could strongly elicit the expressions of hydroxyproline and Col 1A2 over 3 folds and 200 folds, respectively, as compared to the normal control groups (Fig. 4B, C).

HSC activation plays a key role in liver fibrosis at the early phase and activated HSC is accompanied with high expressions of p-Smad2/3 and α-SMA proteins 9101920. Our results mirror this fact as shown in Fig. 1C where both proteins are markedly expressed as compared with the controls. Cirrhosis represents a later stage of progressive scarring in chronic liver disease; it begins with subendothelial or pericentral fibrosis (hepatic fibrosis) and progresses to panlobular fibrosis with nodule formation (cirrhosis) 2. Our study demonstrates that liver TGF-β1 of pPK9a-transferred mice with ZnSO₄ feeding can achieve a substantial increased expression level with fibrosis. However, since our TGF-β1 expression is transient, the fibrotic level does not reach a large scale (panlobular fibrosis) as seen in the long term CCl₄-treated liver [Additional file 1]. Although this CCl₄-induced cirrhosis model is commonly used, its effect is systemic and no attempts are made to clarify the influence of CCl₄ toxicity 3. In this regard, our model is apparently different from the CCl₄ model with respect to TGF-β1; the life of TGF-β1 is transient, dynamic and overexpressed. We also noticed that the transient overexpression of TGF-β1 in the liver leads to an increased deposition of ECM around the vessels as well as along the sinusoids (Fig. 3B). This finding is consistent with the observation described by Ueberham et al. on transgenic animal models 21.

To probe into the down steam effectors of TGF-β1 we refer to the EMSA assay to see if Sp1 molecule specifically is involved in the signaling pathway. TGF-β1 being a strong activator of ECM accumulation stimulates the Col 1A2 gene expression by inducing the binding of a Sp1- and p-Smad2/3-Smad4-containing complex to Col 1A2 upstream promoter element (-330 bp to -286 bp and -271 bp to -255 bp; TGF-β1 responsive element; TbRE) which contains a CAGA box. Since Sp1 is a critical mediator of Col 1A2 expression, we deem it prudent to examine if Sp1 was induced in pPK9a-transferred mice treated with ZnSO₄ and hence performed the supershift assay to confirm the Sp1 and Sp3 binding to TbRE. Fig. 5 shows that the binding activity of Sp1 in liver increased at day 2 and decreased at day 3–5. The pattern of Sp1 binding strongly correlates with the expression levels of TGF-β1 (Fig. 1E). Since it has been reported that Sp1 is required for the early response of Col 1A2 to TGF-β1 and maintenance of the constitutive expression of Col 1A2 20, our results provide direct evidence confirming that our pattern of fibrosis model is early, dynamic and reversible. Moreover, Fig. 3A and 4C show that temporal activation of TGF-β1 and Sp1 is correlated with α-SMA and Col 1A2 expressions, a finding consistent with previous reports 1920.

Mice of Balb/c strain were used. All procedures of animal handling were approved by the Institutional Animal Care and Use Committee of National Cheng Kung University. Eight-week-old mice were used in all experiments and were divided into five groups (n = 21), fed ad libitum standard laboratory feed and water with or without 25 mM ZnSO₄ plus 5% sucrose in the animal facility 22.

TGF-β1 cDNA was constructed in pPK9a vector and was under the regulation of metallothionein promoter. Cys²²³ and Cys²²⁵ in the TGF-β1 pro-peptide were also converted to serine, a mutation that results in dissociation of the pro-peptide and secretion of bioactive TGF-β1 11. It has been found that this mutation does not alter TGF-β1 production but does yield a high proportion of mature 25 kDa dimer which is bioactive without acid activation 1122. pPK9a was a gift from Professor Paturu Kondaiah of the Indian Institute of Science, India.

The plasmid used for administration was purified with a purification kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.

Ten μg of plasmid (pPK9a) were dissolved in 3.0 ml Ringer's solution (NaCl 0.154 M, KCl 5.63 mM, and CaCl₂ 2.25 mM) and injected into the mouse tail vein in a short duration of 5–7 s according to the hydrodynamics-based transfection protocol as described 14. ZnSO₄ (25 mM) was dissolved in the drinking water to activate the metallothionein promoter and stimulate TGF-β1 expression 2223.

Blood was gathered by puncturing into retro-orbital veins with a 27 gauge needle and reserved in a tube for 30 min at 4°C. Serum was separated by centrifugation at 2,640 g for 3 min at 4°C. TGF-β1 levels were determined by enzyme-linked immunosorbent assays (ELISA) method (DuoSet ELISA, R&D Systems, Minneapolis, MN).

RNA was isolated from liver tissue by using Trizol Reagent (GIBCO Life Technologies, Rockville, MD). TGF-β1 mRNA expression was detected by means of RT-PCR with specific primers that distinguish porcine TGF-β1 transcript from that of the mouse endogenous TGF-β1. PCR primers specific for porcine TGF-β1 were: 5'-GAAAGCGGCAACCAAATC-3' and 5'-TGACATCAAAGGACAGCCAC-3'. Additional primers used for RT-PCR of glyceraldehydes-3-phosphate dehydrogenase (GAPDH) gene, were 5'-CCCTTCATTGACCTCAACTAC-3' and 5'-CCACCTTCTTGATGTCATCAT-3'. All RT-PCR reactions were done for 35 cycles 24.

For studying protein expressions of TGF-β1 and α-SMA, liver tissue was homogenized in a RIPA buffer (50 mM Tris-HCl, pH 8; 150 mM NaCl; 1%

Nonidet P-40; 0.1% SDS; 1% Triton X-100 plus protease inhibitors Sigma, St. Louis MO) and centrifuged as described 25; supernatant was taken as a whole-cell lysate. TGF-β1 and α-SMA were electrophoresed under non-reducing condition on a 12% SDS-polyacrylamide gel, transferred by electroblotting to a PVDF membrane, and visualized by immunostaining. Anti-TGF-β1, anti-phospho-Smad 2/3 (Ser433/435-phosphorylated Smad2/3; p-Smad2/3), anti-Smad 2/3, anti-GAPDH and anti-α-SMA antibody (Santa Cruz Bio-technology, Inc., Santa Cruz, CA) were used as the primary antibodies. Secondary antibodies were conjugated with horseradish peroxidase (Bio-Rad Laboratories). The signals were visualized by an enhanced chemiluminescence system (ECL, Amersham).

Hydroxyproline content was determined as reported with slight modification 2526. Briefly, 100 mg of liver sample were hydrolyzed in 6 M HCl at 110°C for 24 h. After centrifugation at 2000 rpm at 48°C for 5 min, 2 ml of supernatant was mixed with 50 ml of 1% phenolphthalein and 8 N KOH to pH7–8. A 5 ml sample was subjected to a spectrophotometer at 560 nm to determine the content of hydroxyproline.

Samples of 1 ml blood were gathered from the retro-orbital plexus of each mouse and immediately centrifuged at 1,300 g at 4°C while plasma was kept at -20°C for liver function tests. ALT levels were measured using a colorimetric analyzer 27 (Dri-Chem 3000, Fuji Photo Film Co, Tokyo, Japan).

Mouse liver tissues were embedded in an optimal cutting temperature (OCT) compound (Miles Inc., Elkhart, IN) and frozen in liquid nitrogen. Five μm-thick cryosections were made by using cryostats (Leica CM 1800, Nussloch, Germany). The sections were fixed with cold acetone and endogenous peroxidase was inhibited by 3% H₂O₂ in phosphate buffered saline (PBS). Then the sections were incubated with 5% blocking serum (normal serum of the species of the secondary antibody). For modeling the negative control sections, the primary antibodies were substituted for the appropriate classes and isotypes of normal immunoglobulins (Igs). Controls for nonspecific binding of the secondary antibody were performed by replacing the solutions of the first step with PBS buffer. TGF-β1 was revealed by using a rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and an anti-rabbit IgG conjugated with Alexa Fluor 594 (Molecular Probes, Eugene, OR). For the detection of α-SMA, a mouse monoclonal antibody was used. Signals were visualized by anti-mouse IgG-HRP, horseradish peroxidase labeled secondary antibody, and 3, 3'-diaminobenzidine substrate (Vector Laboratories, Burlingame, CA). All sections were viewed under a microscope (Leica Mikrosysteme Vertrieb GmbH, Bensheim, Germany) 2125.

Liver specimens were preserved in 4% paraformaldehyde in phosphate-buffered saline and dehydrated in a graded alcohol series. Following xylene treatment, the specimens were embedded in paraffin blocks and cut into 5 μm-thick sections stained with Masson's trichrome as described 2125.

Total RNA was isolated from the liver tissue by TRIZOL reagent (Invitrogen, Carlsbad, CA, USA). RT was performed as described 28. Quantitative real-time PCR was performed with ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). One μg of cDNA was used in each PCR reaction. The housekeeping GAPDH was used as a reference gene for normalization, and H₂O was used as a negative control. The primers for the PCR reactions of Col 1A2 U08020 were: 5'-ACCTGTGTGTTCCCTACTCA-3' and 5'-GACTGTTGCCTTCGCCTC TG-3', the reaction was catalyzed by Taq polymerase (Invitrogen Corp, Carlsbad, CA). SYBR Green I DNA-binding dye generated the fluorescence signals during each of the 35 cycles, in proportion to the quantities of double-stranded DNA (denaturation 15 s at 95°C, annealing 15 s at 56°C and extension 40 s at 72°C). Each sample was analyzed in triplicate. Detection of the PCR products by agarose gel electrophoresis confirmed the homogeneity of the DNA products. Relative quantitation was calculated using the comparative threshold cycle (C_T) method [as described in the User Bulletin #2, ABI PRISM 7700 Sequence Detection System]. Relative quantification of the Col 1A2 transcript was compared to that of the untreated negative control by the following formula: 2−Δ(ΔCT) MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaqabeGadaaakeaacqaIYaGmdaahaaWcbeqaaiabgkHiTiabfs5aejabcIcaOiabfs5aejabboeadnaaBaaameaacqqGubavaeqaaSGaeiykaKcaaaaa@35AA@ and calculated ΔC_{T Col 1A2} = C_{T Col 1A2} - C_{T GAPDH} and Δ(ΔC_T) = ΔC_{T Col 1A2} - ΔC_{T negative control} 29.

The preparation of liver nuclear extracts was based on the method described by Chang and Huang with minor modifications 30. Fresh liver tissue of 0.1 g was homogenized with a Polytron (Kinematica) in 1 ml of buffer A (10 mM HEPES (pH 7.9), 1.5 mM magnesium chloride, 10 mM potassium chloride, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, 2 μg/ml leupeptin, 10 μg/ml aprotinin, 50 mM sodium fluoride, and 1 mM sodium orthovanadate), incubated on ice for 10 min and then gently shaken for 10 s. The pellet of the crude nuclei was collected by centrifugation at 12,000 g for 10 s, resuspended in 300 μl of buffer C (20 mM HEPES (pH 7.9), 25% glycerol, 420 mM sodium chloride, 1.5 mM magnesium chloride, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, 2 μg/ml leupeptin, 10 μg/ml aprotinin, 50 mM sodium fluoride, and 1 mM sodium orthovanadate) by vortex for 15 s, and then incubated on ice for 20 min. After centrifugation at 12,000 g for 2 min, the supernatant containing the nuclear proteins was collected, quantified with BCA Protein Assay Reagent (Pierce), and stored at -70°C in aliquots. For EMSA assay we used the following oligonucleotides: consensus Sp1 (f) 5'-GTT GCG GGG CGG GGC CGA GTG-3' and consensus Sp1 (r) 3'-AAC GCC CCG CCC CGG CTC ACG-5' and labeled the probes with ³²P-dCTP by fill-in method 30.

Results were displayed by means of mean ± SD. Statistical analysis was carried out by F-test (for confirming homogeneity of variances) and two-tailed Student's t-test (for evaluating differences between means). P values lower than 0.05 (*) and 0.01 (**) were considered statistically significant.