Intragastrically Applicated CCl4-Thiopental Sodium Enhanced Lipid Peroxidation and Liver Fibrosis (Cirrhosis) in Rat: Malonedialdehyde as a Parameter of Lipid Peroxidation Correlated with Hydroxyproline as a Parameter of Collagen Synthesis (Deposition)

We investigated the pathogenesis of liver tissue damage during the lipid peroxidation and fibrogenesis with the observation of correlations between the parameters of collagen synthesis (and deposition) and lipid peroxidation in liver fibrosis (cirrhosis) rats. Rats were randomly divided into two groups, normal and CCl4-thiopental sod. intoxicated group. And the one group was treated intragastrically with the mixture of CCl4-thiopental sod. 3 times per week for 3 weeks. The liver tissue and sera were used for the measurement of hydroxyproline (HYP), malonedialdehyde (MDA) and superoxide dismutase (SOD). Biochemical parameters such as aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), total-bilirubin and blood urea nitrogen (BUN) were measured. Additionally, the expression of collagen α1(III) and β-actin mRNA was observed by RT-PCR. The histological change in liver tissue was also observed by Masson’s trichrome and H&E staining. Correlation analysis was carried by Spearman’s rho method. All biochemical parameters except total-bilirubin were significantly higher in the CCl4-thiopental sod. treated group than that of the normal group (p < 0.01). In the CCl4-thiopental sod. treated group, Hyp as a parameter of collagen synthesis (deposition) and MDA as a metabolite of lipid peroxidation, were significantly elevated by 1.98 and 2.11 times higher than that of the normal group (p> 0.001) respectively. The activity of SOD in the CCl4-thiopental sod. treated group is decreased significantly by 44.8% (p> 0.001). And collagen α1(III) mRNA was more expressed in the CCl4-thiopental sod. treated group than that of the normal group. However, the expression of β-actin mRNA is showed similar in both of groups. A good correlation was observed between the content of hyp and MDA concentration (r = 0.70, n = 40) in the two groups. And the correlation between the levels of hyp and SOD (r = −0.71, n = 25) is also reliable. However, no correlation were observed between MDA concentration and SOD (r = −0.40, n = 25) in the two groups. Elevated levels of MDA in CCl4-thiopental sod. treated rats indicated enhancement of lipid peroxidation, which is accompanied by a decrease in SOD activity. Moreover, we could confirm that the parameters of collagen synthesis (and deposition) is in good correlation with the metabolite of lipid peroxidation (MDA) and the lipid peroxidation antagonizing enzyme (SOD). Hence, we propose that ➀ lipid peroxidation and collagen synthesis (and deposition) could be enhanced by intragastrically application of CCl4-thiopental sod. during a short terms. And ➁ the intoxication of CCl4-thiopental sod. could be used for monitoring of lipid peroxidation and collagen synthesis (and depositon) for test of antioxidant and antifibrotic agent.

We investigated the pathogenesis of liver tissue damage during the lipid peroxidation and fibrogenesis with the observation of correlations between the parameters of collagen synthesis (and deposition) and lipid peroxidation in liver fibrosis (cirrhosis) rats. Rats were randomly divided into two groups, normal and CCl4-thiopental sod. intoxicated group. And the one group was treated intragastrically with the mixture of CCl4-thiopental sod. 3 times per week for 3 weeks. The liver tissue and sera were used for the measurement of hydroxyproline (HYP), malonedialdehyde (MDA) and superoxide dismutase (SOD). Biochemical parameters such as aspartate transaminase (AST), alanine transaminase (AL T), alkaline phosphatase (ALP), total-bilirubin and blood urea nitrogen (BUN) were measured. Additionally, the expression of collagen a1(11I) and ~-actin mRNA was observed by RT-PCR. The histological change in liver tissue was also observed by Masson's trichrome and H&E staining. Correlation analysis was carried by Spearman's rho method. All biochemical parameters except total-bilirubin were significantly higher in the CCl4-thiopental sod. treated group than that of the normal group (p < 0.01). In the CCl4-thiopental sod. treated group, Hyp as a parameter of collagen synthesis (deposition) and MDA as a metabolite of lipid peroxidation, were significantly elevated by 1.98 and 2.11 times higher than that of the normal group (p < 0.001) respectively. The activity of SOD in the CCl4-thiopental sod. treated group is decreased significantly by 44.8% (p < 0.001). And collagen a1 (Ill) mRNA was more expressed in the CCl4-thiopental sod. treated group than that of the normal group. However, the expression of ~-actin mRNA is showed similar in both of groups. A good correlation was observed between the content of hyp and MDA concentration (r = 0.70, n = 40) in the two groups. And the correlation between the levels of hyp and SOD (r = -0.71, n = 25) is also reliable. However, no correlation were observed between MDA concentration and SOD (r = -0.40, n = 25) in the two groups. Elevated levels of MDA in CCl4-thiopental sod. treated rats indicated enhancement of lipid peroxidation, which is accompanied by a decrease in SOD activity. Moreover, we could confirm that the parameters of collagen synthesis (and deposition) is in good correlation with the metabolite of lipid peroxidation (MDA) and the lipid peroxidation

INTRODUCTION
The commonly used hepatotoxic agent is CCI4, which transforms into the trichloromethyl free radical (CCI3°) by cytochrome P-450 and readily reacts with O2 to form trichloromethylperoxy radical (CCI 3 0 2°: Parola et a/., 1996). Free radicals and organic radical intermediates can enhance the process of lipid peroxidation which destroys unsaturated fatty acids in cellular membranes (Schuppan, 1990), and produce toxic and reactive aldehyde metabolites such as malonedialdehyde (MDA) and HNE (4-hydroxynonenal: Poli and Parola, 1997). The other hand one of the famous reactive oxygen species (ROS) is superoxide that catalyzes the synthesis of hydrogen peroxide and oxygen by superoxide dismutase (SOD), is a living cell protect enzyme.
Except for gene expression of collagen a1 (Ill), as a marker of collagen synthesis, the best measurable parameter in experimental liver tissue is the hydroxyproline (hyp) content. Hyp comprises approximately 10-12% of the total amino acid in a collagen molecule. The increasing level of hyp in liver is correlated with procollagen type III peptide (Slater, 1994) or MDNHNE (Esterbauer et a/., 1992;Cheeseman et al., 1985) in cholestasis and alcohol induced liver disease. The major pathogenesis of tissue damage is characterized by enhanced lipid peroxidation and deranged antioxidant defensive system (Esterbauer et al., 1992). The tissue fibrosis can be defined by excessive fibrogenesis (collagen synthesis) and progressive accumulation of collagens ,component of extracellular matrix proteins in various liver disease (Ruwart et al., 1989;Kamimura et a/., 1992). But the studies of experimental liver fibrosis (cirrhosis) and manifest reports about the parameters lipid peroxidation and fibrogenesis in short term CCl 4 -intoxication are not exist. In this report, we investigated the pathogenesis of lipid peroxidation and collagen synthesis (and deposition) with analysis correlations of parameters in normal and CCI4 intoxicated rats.
Induction of liver fibrosis (cirrhosis). Female Sprague-Dawley rats (130 to 140 g) were randomly divided into 2 groups (Normal, CCl 4 -thiopental sod. treated group) and having access to the lab chow and water under a light/dark rhythm. For the development of liver fibrosis (cirrhosis), the rats were treated intragastrically with CCl 4 -thiopental sod. mixture (0.6 mltrat, diluted 1 : 1 in olive oil containing thiopental sod. at a concentration of 3.6 mg/rat) three times/week for 3 weeks. The change of body weight was monitored weekly in all rats.
Experimental sampling. Immediately after the sacrifice, blood was collected and centrifuged at 1,500 xg for 10 min at 4°C and the serum was kept at -20°C until use. Liver was promptly dissected and the pieces of liver were fixed in buffered 4% formaldehyde and used for the tissue staining. The rest of liver was immediately stored at -80°C for the determination of hyp, MDA and SOD and frozen in liquid nitrogen for the observation of collagen a1(11I) mRNA and ~-actin mRNA expression by RT-PCR.

Measurement of HYP.
Hydroxyproline was measured by modification of Jamall's method (Jamall et a/., 1981). 0.2 g wet liver tissue was homogenized in 4 ml 6 N HCI and then hydrolyzed at 110°C for 12 hr. 50 J. . l 1 of filtered samples in duplicate were dried, and 1.2 ml of 50% isopropanol added to dissolve. 200 J. . l 1 of chloramine-T solution was added to oxidize and incubated 10 min at room temperature. 1.0 ml of Ehrlich's reagent as a chromogen was used. The mixture of all samples was incubated for 90 min at 50°C and cooled at room temperature. The absorbance of sample was read at 558 nm (Shimadzu UV-1202). The content of hyp was determined from a standard curve generated from known quantities of trans-4-hydroxyproline (0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.5 and 2.0 fl9/50 fll) which had been hydrolysed as described above.
Determination of MDA. The concentration of MDA was determined as a lipid peroxide metabolite by the method of Okawa et al. (1979). 0.25 g liver tissue was homogenized in 1.75 ml of 1.15% KCI. 100 fll of 0.2% SDS were added into 200 fll of homogenates and different diluted malondialdehyde standard solution and incubated for 10 min at room temperature. Thereafter 750 fll of 20% acetic acid (pH 3.5), 750 fll 0.8% thiobarbiturate and 200 fll distilled water were added and incubated for 30 min at 95°C. After cooling on ice, 2 ml of butanol was added into the mixture and centrifuged at 1500 xg for 5 min. The organic layer was used to measure the absorbance by spectrophotometer (Shimadzu UV-1202) at 532 nm. The results were expressed as flmol of MDAlg liver.
Assay of SOD activity. 0.1 g liver tissue was homogenized in 2.0 ml 50 mM Tris buffer (pH 7.6) containing 0.1 mM EDTA and centrifuged at 3500 xg for 10 min at 4°C. The supernatant was directly extracted with a chloroform and ethanol mixture (37.5 : 62.5 v/v), and the suspension was centrifuged at 200 xg for 10 min at 4°C. The supernatant was used for determination of SOD by the modified method of Okado- Matsumoto et al. (2001). 50 fll of diluted sample solution and diluted SOD standard solutions were added into a mixture with 1.25 ml of 50 mM sodium phosphate buffer (pH 8.0), 50 fll each of 3 mM EDTA, 3 mM xanthine and 0.75 mM XTT solution. The reaction was initiated by the addition of 50 fll of 200 mU/ml xanthine oxidase solution. After 10, 20, 30 min incubation at 30°C in a water bath, the absorbance change was measured at 470 nm by spectrophotometer. The results were plotted as IU SOD/0.1 g liver.
Collagen a1 (11/) and {:J-actin mRNA expression by RT-PCR. Total RNA was extracted from liver tissue by using the TRlzol reagent. About 2 fl9 of total RNA were reverse-transcribed into complementary DNA (cDNA). A set of primers (Kauschke et aI., 1999) for the collagen a1 (Ill) and ~-actin gene which give an amplified fragment of 295 bp and 250 bp, resp. were used. The reaction mixture for the PCR consisted of 5 fll of ten-fold PCR buffer, 1.5 fll of 50 mM MgCI2, 1.0 fll of 10 mM dNTP, 35 fll of DEPC H20, 0.5 fll of Taq. polymerase, 1 fll each of a sense (CGC GGG TAC CCT ATG GCA ATC CTG ATC TTC CTG A) and antisense primers (CGC GM GCT TCA TGG CCT GTG TTT GA) of collagen III and ~-actin, 2 fll of synthesized cDNA solution, providing a total volume of 50 fll. After incubation at 94°C for 5 min, the reaction mixture were denatured at 94°C, primers were annealed at 58°C, and DNA synthesis took place at 72°C (each step 45 sec, 30 cycles, Eppendorf, Master cycler gradient, USA). At the end an additional extension time of 5 min at 92°C was performed. Subsequently, the reaction was paused at 4°C. Reaction mixture and 50 bp DNA ladder as DNA size markers were resolved by electrophoresis on 2.0% agarose gel with TBE buffer for ethidium bromide staining. Band intensities of amplified ~-actin and collagen a1(11I) cDNAs were compared after visualization on a UV transilluminator.
Histological procedures. For histological examination, liver specimens were fixed in 4% buffered formaldehyde, and embedded in paraffin and stained with hematoxylin-eosin and Masson's trichrome. The histological changes such as focal necrosis, lipid droplets, inflammation cell and collagen fibers were observed in portal triads and stroma.
Statistical analysis. The significance of all results was tested with Student's t -test and expressed as pvalue. Data show the mean ± standard deviations. Correlation-analyses of each parameter was also evaluated by Spearman's rho method.

RESULTS
Change of liver weight and the ratio of liver weight/body weight Table 1 shows decreased body weight, significantly higher liver weight and the ratio of   Significantly different from normal group (p < 0.005), AC: CCl4-thiopental sod. treated group, n: number of rats. liver weight/body in CCl 4 -thiopental sod. treated rats than that of normal rats (p < 0.005).
Clinical biochemical parameters. Standard liver function tests were analyzed for monitoring of necrosis and liver damage. Significantly increased value of AST and ALT were observed in CCl 4 -thiopental sod. treated rats (p < 0.001, Fig. 1 a). In addition, the levels of BUN and ALP were Significantly greater in CCl4-thiopental sod. treated rats than that of normal rats (p < 0.05-0.001, Fig. 1 a) by 4.44 and 4.88 times. Total-bilirubin in CCl 4 -thiopental sod. treated rats was higher than that of normal group, but showed no significance (Fig. 1 b).
Collagen deposition in liver tissue. The content of hyp in liver tissue is a parameter of collagen deposition and collagen imbalance. The value of hyp is significantly elevated (1.98 times) in the CCl 4 -thiopental sod. treated group compared to normal group (p < 0.001, Table 2).
Lipid peroxidation-derived aldehyde. Activated lipid peroxidation in liver results in liver cell damage and enhanced fibrogenesis. The concentration of MDA as a lipid peroxidation metabolite was 2.11 times higher in CCl 4 -thiopental sod. treated group than that of normal group (p < 0.001, Table 2).
SOD activity. One of the major protective enzyme in living cell is SOD. The concentration of SOD is signif-icantly decreased (48.2%) in the CCl 4 -thiopental sod. treated group compared to the normal group (p < 0.001, Table 2).

Collagen a1(1I1) and {J-actin mRNA expression.
The expression of ~-actin mRNA which was used as a housekeeping gene was almost similar in both group (Fig. 5). However collagen a1(1I1) mRNA mRNA was more expressed in the CCl4-thiopental sod. treated group than that of normal group (p < 0.001, Fig. 2).
Relationships among hyp, MDA and SOD. The content of hyp were correlated with MDA concentration (r = 0.70, n = 40) or SOD (r = -0.71, n = 25), while very weak negatively correlation were observed between MDA and SOD (r = -0.40, n = 25) as shown in Fig. 3. lagen fiber in portal triads, lysis of cytoplasma and crashed down nuclei were observed in CCl 4 -thiopental sod. treated group by staining with Masson's trichrome. Moreover, collagen fibers bridged one portal and neighbor portal area and appearance of inflammatory cells were observed in CCl 4 -thiopental sod. treated group (Fig. 4) .

DISCUSSION
Oxidative stress has been shown to enhance lipid peroxidation that has been indicated relationship between chronic liver damage and fibrosis (Cheeseman et aI., 1985;MacDonald et al., 2001;Vengerovskii et aI., 1996). In this study, the pathogenesis of liver tissue damage was investigated by the observation of correlation between the parameters of collagen synthesis (and deposition) and lipid peroxidation in liver fibrosis (cirrhosis) induced rats treated a short term with CCI 4 -thiopental sod. Our study showed that the value of MDA as a product of lipid peroxidation in liver tissue and the level of AST and ALT in sera of CCl4-thiopental sod. treated group as a biochemical parameter of necrosis were significantly increased (p < 0.005, Fig. 1 a). Surveying the studies of the past, a long term CCl 4 -intoxication could be induced lipid peroxidation and liver damage, and thereby appeared increased levels of MDA, AST and ALT (Gasso et al., 1996;Hernandez-Munoz et al., 1997), but the change of SOD activity is highly disputed (Hernandez-Munoz et aI., Cabre et al., 2000). Additionally, increased product of lipid peroxidation (MDA, HNE) were reported not only in the plasma of patient with decompensated liver cirrhosis and in hepatitis C liver tissue (Chen et aI., 1997;Paradis et aI., 1997), as well as in the liver tissue of animals with ethanol, dimethylnitrosamine, or iron overload induced liver damage (Bacon and Britton, 1990;Niemelae et al., 1995;Vendemiale et aI., 2001). Our results shows enhanced lipid peroxidation and necrosis were developed by a short term CCI4-thiopental sod. intoxication.
Nadkarni and D'Souza reported that only the concentration of SOD, which had been shown to antagonize lipid peroxidation, in CCl 4 -phenobarbitone treated rats. However no increasing in hepatic lipid peroxidation metabolite (MDA) was observed. These results suggests that in cirrhotic livers cell damage are resulted by the direct attack of oxygen free-radicals (Nadkarni and D'Souze, 1998). Henandez-Mundez et al. reported that activity of SOD after 4 and 8 weeks CCl 4 -treatment was similar in the normal and CCl 4 -intoxificated group (Hernandez-Munoz et al., 1997).
In the current study, the concentration of SOD in the CCl 4 -thiopental sod. treated group was decreased by c) d) 44.8%, showing a similar trend as in Nadkarni's report (Nadkarni and D'Souza, 1998), on the other hand the lipid peroxidation product (MDA) was increased 2.11 times ( Table 2). The differential results can be bring down to various influence factors. The administration method of CCI4 could be the one of the factors. Nadkarni and D'Souza used CCI 4 --gassing plus phenobarbitone in drinking water. In our study, we used a mixture of CClcthiopental sod. intragastrically.
From this argument, our data could be indicated that liver damage may be induced by direct attack of free radical and thereby lipid peroxidation stimulated and the balance of antioxidant defensive system might be broken. Also we observed that the expression of collagen a1(11I) mRNN~-actin mRNA was increased in CCI4-thiopental sod. treated group than that of normal group (Fig. 2). Similar result was reported that increased in the expression of collagen a1(1I1) mRNA is an indicator of collagen synthesis by quantitative RT-PCR in dimethylnitrosamine induced liver fibrosis (Shiba et al., 1998) with CCI4-induced liver fibrosis (Kauschke et al., 1999). And increased concentration of MDA and expression of collagen a1(1I1) mRNA were related with activation of HSC (Lee et a/., 1995;Maher et al., 1994).
Our data demonstrated that lipid peroxidation could play a key role in fibrogenesis of liver disease as reported (MacDonald et a/., 2001;Ljubuncic et a/., 2000;Rhoden et a/., 2000). It might act as a link between tissue injury and liver fibrosis by modulating collagen gene expression (MacDonald et a/., 2001;Ljubuncic et al., 2000). A lot of research about good correlation between the parameters of lipid peroxidation, fibrogenesis, and collagen deposition presented e. f. MDA correlated with the score of fibrosis in liver tissue of patient with hepatitis C (Paradis et al., 1997). In gluthation peroxidase in sera of CCl 4 -treated rats was also reported (r = -OA7, P < 0.001) (Cabre et a/., 2000).
Our data showed good correlation between hyp and MDA (r = 0.70, n = 40) in liver tissue of normal and CCl 4 -thiopental sod. treated. The SOD activity correlated negatively with hyp (r = -0.71, n = 25) and with MDA (r = -OAO, n = 25). As a result, the treatment with CCl 4 -thiopental sod. enhanced free radical generation, stimulated excessive lipid peroxidation and enhanced collagen synthesis and deposition.
It was reported in minipig with ethanol-induced liver disease that products of ethanol metabolism such as acetaldehyde and the metabolite of lipid peroxidation (MDA) appeared to precede necrosis and fibrosis (Niemelae et al., 1995). Similar results were observed in our study. Thus the values of ALT and AST as an indicator of necrosis were higher in CCl4-thiopental sod. treated group than that of the normal group (p < 0.05-0.001, Fig. 1). The necrosis and fibrosis of liver tissue were observed by Masson's trichrome staining in CCI 4thiopental sod. treated group (Fig. 4). In addition, a significantly higher liver weight and enhanced ratio of liver/ body weight as a sign of hepatomegaly were induced by CCl 4 -thiopental sod. intoxication (p < 0.001, Table 1).
In conclusion, elevated levels of MDA in CCI 4 -thiopental sod. treated rats indicated enhancement of lipid peroxidation, which is accompanied by a decrease in SOD activity. Moreover, we could confirm that the parameters of collagen synthesis (and deposition) is in good correlation with the metabolite of lipid peroxidation (MDA) and the lipid peroxidation antagonizing enzyme (SOD). Hence, we propose that lipid peroxidation and collagen synthesis (and deposition) could be enhanced by intragastrically application of CCl 4 -thiopental sod. during a short terms. And the intoxication of CCI 4 -thiopental sod. could be used for monitoring of lipid peroxidation and collagen synthesis (and depositon) for test of antioxidant and antifibrotic agent.