Original Paper Jourr~[~
Biomedical Science
Received:August9, 2002 Accepted:November7, 2002
J Biomed Sci 2003;10:170-178 DOI: 10.1159/000068715
Oxidative-Stress-Related Changes in the Livers of Bile-Duct-Ligated Rats Yi-Tsau Huang a Yi-Chao Hsu a Chi-Jen Chen a Chien-Tzu Liu a Yau-Huei Wei b alnstitute of Traditional Medicine, School of Medicine, and bDepartment of Biochemistry, School of Life Science, National Yang-Ming University, Taipei, Taiwan, ROC
Key Words Fibrosis. Malondialdehyde • 8-Hydroxy-2'deoxyguanosine • Transforming growth factor-J31. Collagen • Mitochondrial electron transport chain
Abstract The role of reactive oxygen species in liver fibrogenesis is not yet clarified. The aim of this study was to investigate oxidative-stress-related changes in cirrhotic rats. Cirrhosis was induced by bile duct ligation in SpragueDawley rats. Plasma malondialdehyde (MDA), hepatic 8hydroxy-2'-deoxyguanosine (8-OHdG), hepatic mitochondrial respiratory functions and gene transcripts were measured at 2 and 4 weeks after surgery in bileduct-ligated (BDL) and sham-operated-operated rats. The results showed progressive increases in the levels of plasma MDA, hepatic 8-OHdG and procollagen 1 and II1 mRNA expression, and progressive impairment of hepatic mitochondrial respiratory function in BDL rats at 2 and 4 weeks after ligation compared with sham-operated rats. Moreover, at 4 weeks after ligation, BDL rats exhibited reduced plasma glutathione and vitamin E levels, impaired hepatic mitochondrial electron transport enzyme activities and oxidative phosphorylation function. In addition, hepatic mRNA expression of transforming
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growth factor-J31 was increased. Hepatomegaly, abnormal plasma alanine transaminase and aspartate transaminase levels, and portal hypertension were noted in BDL rats. Our results suggest that bile duct ligation in the rat induces mitochondrial dysfunction and biochemical and molecular changes related to oxidative stress in the liver. Copyright© 2003NationalScienceCouncil,ROCand S. KargerAG, Basel
Introduction
Progressive hepatic fibrosis can lead to cirrhosis with portal hypertension and hyperdynamic circulation [13]. The initiating events for hepatic fibrosis include viral hepatitis, alcoholism, and bile duct obstruction [ t 3]. Hepatic fibrosis has been conceptually viewed as a chronic 'response-to-injury' wound-healing process, which is dynamic and reversible [10, 36]. There are several hallmarks of hepatic fibrosis: inflammation, recruitment and local proliferation of myofibroblast-like cells, and deposition as well as remodeling of the extracellular matrix [10, 36]. Continuous processes such as accumulation of fibrilforming matrix, loss of hepatocytes, and reduced sinosoidal cell porosity contribute to impaired hepatic function, and decreased sinosoidal transport permeability, as well
Yi-Tsau Huang Institute of Traditional Medicine, School of Medicine National Yang-Ming University, 155, Section 2, Li-Nong Street Taipei 112, Taiwan (ROC) Tel. +886 2 28267179, Fax +886 2 28225044, F~Mail
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as portal hypertension [10, 36, 39]. Activation of hepatic myofibroblast-like (i.e., steUate) cells pIays a very crucial role in the progression of hepatic fibrosis, including deposition of extracellular matrix and secretion of growth factors and cytokines [10, 36, 39]. Stellate cell activation denotes phenotype transformation of quiescent, vitaminA-rich cells into proliferative, contractile and fibrogenic cells [t0, 36]. It has been suggested that factors such as transforming growth factorq31 (TGFq31), endothelin-1, and lipid peroxides are involved in hepatic fibrogenesis [10, 36, 39]. Mitochondria are the major intracellular source of reactive oxygen species (ROS) in animals [14, 31]. Excess ROS production and/or defective cellular antioxidant systems have been implicated in aging processes and several degenerative conditions [ 14, 31]. When cellular production of ROS overwhelms its antioxidant capacity, damage to cellular macromolecules such as lipids, proteins and DNA may ensue [48]. In acute hepatic injury, ROS in liver tissue can be derived from Kupffer cells, neutrophiIs, or hepatocytes (e.g., in the cytochrome P4502El-dependent metabolic activation of toxic chemicals such as alcohol, carbon tetrachloride (CC14), and acetaminophen) [19, 21]. The tissue levels of malondialdehyde (MDA) and 8hydroxy-2'-deoxyguanosine (8-OHdG) content in DNA have been reported as markers for oxidative damage in pathophysiological conditions such as inflammation, aging and cancer [14, 15, 42]. The role and mechanisms of ROS and tissue antioxidant systems in chronic liver injury and fibrogenesis have not yet been clarified [10, 36, 39]. The present study investigated the oxidative-stress-related changes in a rat model of hepatic fibrosis, i.e., bile-duct-ligated (BDL) rats, which have portal hypertension [9, 29, 57] and excessive bile acids in the blood [57]. Materials and M e t h o d s BLD Rats
rats were examined for evolutional changes in the parameters listed below. On the day of measurement, each rat first underwent hemodynamic measurement under anesthesia. Venous blood was then withdrawn, and thereafter the rat was sacrificed by KC1 injection to remove the liver for homogenization and biochemical analysis.
Portal Pressure Measurement For each group of rats, hemodynamic studies were performed under ketamine anesthesia (100 mg/kg intraperitoneally) after overnight fasting. Mean arterial pressure (MAP) and portal venous pressure (PVP) were determined by direct cannulation of the right femoral artery and the ileocolic vein, respectively, with PE 50-gauge tubing. Changes in pressures and heart rate were monitored with a polygraph (RS 3400, Gould, Valley View, Ohio, USA) via strain gauge transducers (P23XL, Viggo-Spectramed, Oxnard, Calif., USA). Readings were taken after a steady baseline was reached for 30 min.
Measurement of Plasma MDA Plasma lipid peroxides, as measured by liquid-chromatographic separation of MDA-thiobarbituric acid (TBA) adduct, were assayed primarily according the method of Wong et al. [54]. Briefly, an aliquot of 50 gl of plasma was boiled with 250 gl of phosphoric acid (H3PO4) at 100 °C for 60 min to hydrolyze lipoperoxides. After centrifugation at 16,000 g for 15 min, the supernatant was collected and complexed with TBA. Plasma proteins were precipitated with methanol and removed after centrifugation. The protein-free extract was fractionated by high-pressure liquid chromatography (HPLC) on a column of C ls silica gel to separate the MDA-TBA adduct from interfering chromogens. The MDA-TBA adduct was eluted from the column with methanol/phosphate buffer (pH 6.8) and quantified spectrophotometrically at 532 nm. Plasma lipoperoxide concentrations were calculated by reference to a calibration curve constructed by using 1,1,3,3-tetramethoxypropane as standard.
Biochemical Analysis of Plasma At the end of hemodynamic recording, blood samples were collected (6 ml each from the femoral vein) and immediately centrifuged at 1,300 g at 4 ° C, and plasma was kept at -20 ° C for liver and renal function tests. Aspartate transaminase (AST), alanine transaminase (ALT), and creatinine were measured by standard laboratory methods on an automatic analyzer (Hitachi 736-60, Tokyo, Japan), as described previously [ 18]. Glutathione levels were measured spectrophotometrically at 412 nm with the addition of glutathione reductase, while vitamin E levels were measured by HPLC [47].
8-OHdG Assay
Common bile duct ligation was performed in male Sprague-Dawley rats (250-300 g) to induce hepatic fibrosis and portal hypertension [24] as previously described [18]. A double ligation of the bile duct was performed in rats under anesthesia by a proximal ligature around the bile duct in the hilus of the liver and by a distal ligature close to its entry into the duodenum. A cut was then made between ligatures. On sham-operated rats, the bile duct was mobilized but not ligated. Rats were maintained on a standard rat pellet diet and tap water ad libitum. Animal studies were approved by the Animal Experiment Committee of the University and conducted humanely, in accordance with the Guide for the Care and Use of Laboratory Animals (National Academic Press, USA, 1996). Two weeks (group 1) or 4 weeks (group 2) after bile duct ligation or sham operation, the
8-OHdG levels in DNA from liver tissues were measured primarily according to the method of Helbock et al. [16]. Briefly, fresh rat liver (0.8 g) was homogenized with 10 m M Tris-HC1 (pH 7.4) buffer containing 1 m M EDTA, 0.08% butylated hydroxytoluene, 100 gg/ml RNase, 50 lxg/ml proteinase K and 0.5 % sodium dodecyl sulfate in an ice bath. The mixture was incubated in an oven at 56 ° C for 8 h to degrade proteins and then extracted sequentially with equal volumes of saturated phenol, phenol:chloroform (1:1, v/v), and chloroform:isoamyl alcohol (24:1, v/v). The supernatant was collected after centrifugation at 900 g and, with addtion of 3 M sodium acetate and isopropranol, DNA was precipitated after overnight incubation. The precipitate was collected after ultracentrifugation at 12,000 g and rinsed twice with ethanol under argon flow. DNA was digested in
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10 mM Tris-HC1 (pH 7.4) containing 0.1 mM desferoxamine mesylate, 20 U DNase I, and 0.1 M MgC12. To the solution, 1 M sodium acetate, 0.1 M ZnSO4 and 2 U P1 nuclease were added, followed by incubation with 2 U alkaline phosphatase in 50 mM Tris-HC1 (pH 8.0) at 37 °C for 30 min. After centrifugation at 12,000 g, the supernatant was collected and filtered. The sample was injected into and fi-actionated by an HPLC column with a mobile phase (pH 5.5) composed of 12.5 mM citric acid, 25 m M sodium acetate, 10 mM acetic acid and 6o/0 methanol. The flow rate was adjusted to 0.8 ml/min. The 8-OHdG in the sample was quantified by an eletrochemical detector (Bioanalytical Systems, West Lafayette, Ind., USA) as established in our laboratory [47].
mixture was quickly applied into a Sephadex G-25 column to separate ferrocytochrome c from Na2S204. The assays of mitochondrial respiratory enzyme activities were performed according the method of Zheng et al. [56] and have been established and validated in our laboratory [49]. We used them to measure the electron transport activities of mitochondria from BDL and sham-operated rats. In this study, we did not determine the tissue concentration of NADH by measuring the absorbance at 340 rim, because it does not reflect the activity of any of the respiratory enzymes that we studied. Protein content was determined according to Bradford [2], with bovine serum albumin as the standard.
Analysis of Transcriptsfor TGF-fll and Procoltagen Genes Assay of Mitochondrial Respiratory ControlRatio (RCR) Assays of the RCR and electron transport activities of mitochondria were performed according to the method of Vereesi et al. [52], and have been established in our laboratory [49]. Briefly, livers were homogenized immediately in ice-cold SEH buffer (0.25 M sucrose, 0.5 mMEDTA and 3 n~14HEPES, pH 7.2). The mitochondrial pellet was obtained by low-speed centrifugation (800 g) of the homogenate, tbllowed by high-speed centrifugation (9,500 g) of the supenmtant. The pellet was washed twice with the SEH buffer. About 25-30 mg of mitochondrial protein was obtained from 1 g wet weight of liver. Succinate-supported and glutamate-malate-supported respiration rates of the isolated mitochondria were measured in a Gilson 5/6 oxygraph (Gilson Medical Electronics, Middleton, Wisc., USA), which was equipped with a Clark-type oxygen electrode in a waterjacketed chamber maintained at 25 °C. Freshly isolated mitochondria were incubated in the chamber containing 1.6 ml of assay buffer (125 IrL~fsucrose, 20 ns~/cytochrome c, 6 rm/k/MgC12, 50 m M KC1, 5 m M HEPES, 10 mM NaH2PO4-K2HPO4 buffer). Succinate was injected at a final concentration of 20 m M as substrate to initiate state 4 respiration, which was recorded by a strip chart recorder for 3 min. An aliquot of ADP (400 nmol) was added to initiate state 3 respiration. Respiration returned to state 4 after depletion of ADP. The respirator?' control ratio was expressed as the ratio of the respiration rate of state 3 to that of state 4. The oxidative phosphorylation function was measured as the ADP/O ratio (the amount of ADP added divided by the amount of oxygen consumed in state 3), according to standard procedures [49, 52].
Assay ofMitochondriaI Electron TransportChain Enzyme Activities The enzyme activities of the mitochondrial electron transport chain were measured using a Beckman UV/visible spectrophotometer. The NADH cytochrome c reductase (NCCR) activity assay was carried out by following the reduction of exogenous oxidized cytochrome c, which was monitored by recording the change in absorbance at 550 nm after addition of freshly prepared 13-NADH to the submitochondrial particles prepared by sonication and ultracentrifugation of mitochondria [49]. The succinate cytochrome c reductase (SCCR) activity assay was carried out by following the reduction of exogenous oxidized cytochrome c, which was monitored by recording the change in absorbance at 550 nm. The cytochrome c oxidase (CCO) activity assay was carried out by following the oxidation of exogenous reduced cytochrome c and recording the change in absorbanee at 550 nm after addition of ferrocytochrome c into the assay mixture containing the mitochondrial suspension. Ferrocytochrome c was prepared from 1 Moxidized cytochrome c by reduction with an excess amount of Na2S204 at 25 °C for 5 rain. After reduction, the
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Expression levels of transcripts of TGF-131 and procollagen genes in the liver were analyzed by RT-PCR coupled with densitometric analysis of the ethidium-bromide-stained PCR products after agarose gel electrophoresis. Total RNA was isolated by the method of Chomczynski and Sacchi [5]. Briefly, liver tissue was homogenized with a mixture of guanidium thiocyanate-phenol-chloroform to extract RNA. The primer sequences for PCR amplification (TGF-[31 forward primer: 5'-TAT AGC AAC AAT TCC TGG CG-3', reverse primer: 5'-TGC TGT CAC AGG AGC AGTG-3"; procollagen I forward primer: 5'-TAC TAC CGG GCC GAT GAT GC-3', reverse primer: 5'-TCC TTG GGG TTC G G G CTG ATG TA-3'; procollagen III forward primer: 5'-CCC CTG GTC CCT GCT GTGG-3', reverse primer: 5'-GAG GCC CGG CTG GAA AGAA-3') were designed according to the sequences published by Wasser et al. [53]. The identities of the resultant PCR products were confirmed by sequence analysis. A standard curve was first established to demonstrate the range over which the PCR product yield provided a reliable measure of mRNA input. An aliquot of 0.5 gg total RNA from each liver sample was subjected to reverse transcription by using Moloney leukemia virus (MMuLV) reverse transcriptase (Gibco/Life Technologies, Gaithersburg, Md., USA) in a 30-I~1reaction mixture. The levels of all transcripts were quantified using the glyceraldehyde-3phosphate dehydrogenase (G3PDH) mRNA level in the same tissue as an internal standard [53]. The PCR conditions for measuring TGF-131, procollagen I and III, and G3PDH genes were optimized to ensure that amplification of each gene was in the exponential phase. The number of cycles was optimized at 30 after examining the yield of PCR products at a range of 10-40 cycles. The following thermal cycle was used: denaturation at 94°C for 5 min, followed by three temperatm'e cycles: 94°C for 30 s, 58°C for 30 s and 72°C for 30 s, followed by a final extension step at 72 ° C for 7 rain, conducted in a GeneAmp PCR System (9700 Base Unit, PE Applied Biosystems, Foster City, Calif., USA). To prevent contamination of genomic DNA, all RNA samples were subjected to digestion by DNase (Promega Co., Madison, Wisc., USA), and checked by 30 cycles of PCR to confirm the absence of contaminated DNA. All PCR were carried out in 20-1al volumes, with 200 pmol &each primer and 0.5 units of Taq DNA polymerase. The PCR products were size-fractionated on an agarose gel, visualized by ethidium bromide staining and photographed by a Kodak DC120 digital camera, scanned by a Kodak Electrophoresis Documentation and Analysis System (EDAS 120), and quantified using Kodak Image Analysis Software (Rochester, N.Y., USA).
Data Analysis Data are expressed as mean + SEM. One-way analysis of variance (ANOVA) was used for comparison of hemodynamic parame-
Huang/Hsu/Chen/Liu/Wei
ters. A non-parametric method (the Dunn procedure under the Kruskal-Wallis test) was used for multiple pairwise comparison between groups for the histological grades of fibrosis. Significance was determined at p < 0.05.
Results
General Features BDL rats at 2 and 4 weeks after surgery showed progressive jaundice, hepatomegaly (liver weight (g)/100 g body weight = 7.17 -+ 0.29 for BDL rats vs. 3.26 _+ 0.19 for sham-operated rats, p < 0.01), splenomegaly, and ruesenteric edema. Variable degrees of ascites were observed in BDL rats at 4 weeks [18, 24]. The body weights of BDL and sham-operated rats were similar at both 2 weeks (331 + 13 vs. 374 + 9 g) and 4 weeks (400 + 13 vs. 423 + 13 g) after surgery.
Portal Venous Pressure The BDL rats at 4 weeks showed significantly increased PVP (15.3 -+ 1.1 vs. 8.3 + 0.8 mm Hg, p < 0.01) compared to the sham-operated rats. However, neither MAP (91.8 _+ 3.8 vs. 91.5 +_ 3.7 mm Hg) nor heart rate (264 -+ 17 vs. 302 + 16 beats/min) was different between BDL and sham-operated rats.
Plasma MDA Levels Plasma MDA levels in BDL rats were significantly increased compared with sham-operated rats at both 2 weeks (37.5 -+ 1.9 vs. 6.1 _+ 0.4 nmol/ml, p < 0.01) and 4 weeks (46.8 + 2.2 vs. 6.6 -+ 0.6 nmol/ml, p < 0.0I) alter surgery. The MDA levels of BDL rats at 4 weeks were significantly higher those at 2 weeks, suggesting that the extent of lipid peroxidation increased with progressive fibrosis.
Plasma Biochemistry
8-OHdG Levels BDL rats at 4 weeks showed significantly increased levels of hepatic 8-OHdG levels (8-OHdG/105 dG ratio: 21.9 + 3.4 vs. 12.7 + 0.6, p < 0.05). However, the 8O H d G levels were not significantly different between BDL and sham-operated rats at 2 weeks (8-OHdG/105 dG ratio: 15.4 -+ 0.9 vs. 12.7 + 1.5). The 8-OHdG levels of BDL rats at 4 weeks were significantly higher than that at 2 weeks (8-OHdG/105 dG ratio: 21.9 _+ 3.4 vs. 15.4 + 0.9, p < 0.05), suggesting that the extent of DNA oxidation increased with progressive fibrosis.
Mitochondrial R CR There was a progressive decrease in the RCR in BDL rats with succinate as well as glutamate-malate as the substrate compared with sham-operated rats at both 2 weeks and 4 weeks after surgery. The RCRs of succinate-supported respiration for BDL and sham-operated rats were 3.33 + 0.17 vs. 4.18 + 0.15 (p < 0.01) at 2 weeks, and 2.62 + 0.14 vs. 3.73 +- 0.11 (p < 0.01) at 4 weeks. The RCRs of glutamate-malate-supported respiration for BDL and sham-operated rats were 4.50 + 0.30 vs. 5.89 + 0.27 (p < 0.01) at 2 weeks, and 2.80 + 0.24 vs. 4.61 + 0.36 (p < 0.01) at 4 weeks. The ADP/O ratios of succinate-supported and glutamate-malate-supported respiration were also significantly decreased in BDL rats compared with sham-operated rats. The ADP/O ratios of succinate-supported respiration for BDL and sham-operated rats were 1.89 +- 0.05 vs. 2.14 + 0.05 ( p < 0.01) at 2 weeks, and 1.61 + 0.03 vs. 1.98 + 0.04 (p < 0.01) at 4 weeks. The ADP/O ratios of glutamate-malate-supported respiration for BDL and sham-operated rats were 2.84 + 0.08 vs. 3.18 + 0.07 (p = 0.035) at 2 weeks, and 2.72 + 0.08 vs. 3.00 + 0.10 (p < 0.01) at 4 weeks.
Mitochondrial Electron Transport Chain Enzyme Activities
BDL rats at 4 weeks showed significantly increased levels of both ALT (176 + 18 vs. 35 + 8 U/l, p < 0.01) and AST (908 + 68 vs. 187 + 13 U/l, p < 0.01) compared with sham-operated rats, indicating severe hepatic injury [18]. Both glutathione (3.5 +- 0.3 vs. 11.7 _+ 1.3 gmol/ml, p < 0 . 0 0 1 ) andvitamin E(2.1 + t.1 vs. 3.7 + 3.1 gg/ml, p < 0.05) levels were significantly reduced in BDL rats compared with sham-operated rats at 4 weeks. Plasma creatinine levels were similar between BDL and shamoperated rats, suggesting no manifest renal function impairment in BDL rats.
The NCCR activity was significantly decreased in BDL rats compared with sham-operated rats (142 + 14 vs. 189 + 9 nmol/min/mg protein, p = 0.02) at 4 weeks. However, the activities between BDL and sham-operated rats at 2 weeks were not significantly different (t77 + 7 vs. 210 + 21 nmol/min/mg protein, p = 0.14). Similarly, the SCCR activity was significantly decreased in BDL rats compared with sham-operated rats (32.1 + 4.3 vs.53.4 + 2.5 nmol/min/mg protein, p < 0.0i) at 4 weeks. But the activities between BDL and sham-operated rats at 2 weeks were not significantly different (38.0 + 6.6 vs. 54.7 + 8.9 nmol/min/mg protein, p = 0. i0). In contrast, there
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Fig. 1. Expression of TGF-~ 1 transcript in sham-operated and BDL rats at 2 and 4 weeks after surgery. The RT-PCR amplification products of TGF-~I and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) are visualized. The density of TGF-[31 to G3PDH mRNA levels was analyzed by computerized densitometry and expressed as the TGF-[31:G3PDH ratio. The number of rats in each column is 8. * p < 0.05 vs. sham-operated group.
Fig. 2. Expression of procoUagen I transcript in sham-operated and BDL rats at 2 and 4 weeks after surgery. The RT-PCR amplification products of procollagen I and G3PDH are visualized. The density of procollagen I to G3PDH mRNA levels was analyzed by computerized densitometry and expressed as the pmcollagen I:G3PDH ratio. The number of rats in each column is 8. * p < 0.05 vs. sham-operated group.
was no difference in the CCO activity between BDL and sham-operated rats at either 2 weeks (687 + 54 vs. 719 + 109 nmol/min/mg protein) or 4 weeks (734 + 76 vs. 750 + 50 nmol/min/mg protein).
in both pro-collagen I and pro-collagen III gene transcripts in BDL rats, while TGF-131 m R N A expression was not further increased at 4 weeks compared with that measured at 2 weeks (fig. 1-3).
Analysis of Transcriptsfor TGF-fll and Procollagen Genes There was a significant increase in m R N A expression ofTGF-131 (fig. 1), pro-collagen I (fig. 2), and pro-collagen III (fig. 3) genes relative to G 3 P D H in BDL rats compared with sham-operated rats at both 2 and 4 weeks after surgery. Moreover, there was a time-dependent increase
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Discussion
To our knowledge, the present study was the first to examine alterations in gene expression o f fibrogenesisrelated genes, oxidative D N A damage and mitochondrial respiratory function in the liver of B D L rats. Our results
Huang/Hsu/Chen/Liu/Wei
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Fig. 3. Expression ofprocollagen III transcript in sham-operated and BDL rats at 2 and 4 weeks after surgery. The RT-PCR amplification products of procollagen III and G3PDH are visualized. The density of procollagen III to G3PDH mRNA levels was analyzed by computerized densitometry and expressed as the procollagen III:G3PDH ratio. The number of rats in each column is 8. * p < 0.05 vs. shamoperated group.
showed that the rats at 4 weeks after bile duct ligation exhibited (a) increases in hepatic 8-OHdG levels, (b) increases in hepatic mRNA expression of TGF-[31, and procollagen I and III genes, (c) decreases in hepatic mitochondrial respirator?' control and ADP/O ratios, (d) decreases in hepatic mitochondrial NCCR and SCCR enzyme activities, (e) increases in plasma MDA, ALT and AST levels, (f) decreases in plasma levels of glutathione and vitamin E, and (g) portal hypertension and hepatomegaly. It has been reported that lipid peroxidation induced in hepatic stellate cells increases procollogen I gene expression [37]. Oxidative stress also induces cellular prolifera-
Oxidative-Stress-Related Changes in BDL Rats
tion and type I collagen accumulation in hepatic stellate cells [35, 46]. There are several reports showing that the activation of hepatic stellate cells by TGF-[31 is partially mediated by or associated with increased oxidative stress or the ROS signaling cascade [7, 11]. Thus both TGF-[~I and oxidative stress may mutually reinforce each other in stellate cell activation and hepatic fibrogenesis [8]. Several experimental studies also demonstrated that administration of agents capable of inhibiting lipid peroxidation or scavenging ROS can reduce hepatic fibrosis in treated animals [18, 38, 43, 44, 55]. It has been previously shown that liver mitochondrial functions, including respiratory control and ADP/O ratios, and SCCR enzyme activities are impaired, whereas CCO activity is unchanged in BDL rats [26]. But this previous study only examined the mitochondrial respiratory functions at 5 weeks after ligation, whereas in the present study we examined the changes at both 2 and 4 weeks and employed different study protocols including different substrates (e.g., two different substrates: succinate and glutamate-malate, instead of ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine) for assays of the RCR and ADP/O ratio. It is important to evaluate the RCR and ADP/O ratio using two different substrates as a previous study indicated that these ratios are reduced or unchanged, depending on different substrates given, in CC14-induced cirrhotic rats [25]. In that study, only SCCR (complexes II and III of the electron transport chain) and CCO (complex IV) activities were measured, whereas we further assessed NCCR (complexes I and III) activity in BDL rats. The results of these studies suggest that the mitochondrial electron transport chain function was impaired along complexes I-III but not altered at complex IV. It has been reported that hepatic MDA-TBA adduct levels are increased, and mitochondrial glutathione levels are decreased in BDL rats [28]. In this study, we further observed that the plasma MDA-TBA adduct level was increased, and both glutathione and vitamin E levels were decreased in BDL rats. The results reflected reduced antioxidant capacity in the liver and plasma in BDL rats. In vitro studies have shown that both lipophilic and hydrophobic bile acids can disrupt the mitochondrial electron transport chain and generate lipid peroxides in hepatocytes [27, 45]. This study further demonstrated that rats subject to bile duct ligation for 2 and 4 weeks could have excessive accumulation of bile acids [57] and they exhibited partial, but not complete, impairment of mitochondrial electron transport chain functions in the liver.
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It is well recognized that stellate cells are the key player in hepatic fibrosis and that 'modulation of gene expression through altered transcription' is a critical level of regulation for stellate cell behavior in normal and pathophysiological conditions [8, 10]. Thus, it is of great interest to measure the expression levels ofTGF-131 and pro-collagen I and III genes in BDL rats. A significant increase in TGF131 mRNA expression, albeit at different time points from ours, was reported in liver tissue after bile duct ligation [34]. Increased hepatic TGF-131 mRNA expression has also been documented in cirrhotic patients [4], and in animal models of hepatic fibrosis induced by CC14 [53], dimethlynitrosamine [51] and pig serum [40]. On the other hand, a novel TGF-13 receptor antagonist has been shown to attenuate hepatic fibrosis in BDL rats and inhibit stellate cell activation [12]. The important role of TGF131 in hepatic fibrogenesis has been further demonstrated by the observation that targeted overexpression of TGF131 enhances liver fibrosis under CC14 challenge, whereas TGF-131 knockout mice are less susceptible to fibrogenetic injury [ 17]. Similarly, pro-collagen I mRNA expression in liver was found increased in cirrhotic patients [4] and animal models of hepatic fibrosis induced by CC14 [5 3], dimethylnitrosamine [41], pig serum [40] and bile duct occlusion [20]. In our laboratory, we also observed that there was significantly enhanced immunohistochemical staining for both type I and III collagens in the liver sections from 4week BDL rats (data not shown). As we reported previously [18], the liver sections from BDL rats at this stage of disease show structural changes including disordered architecture with bile duct proliferation and fibrous septa. It has been reported that TGF-131 stimulates collagen synthesis and pro-collagen I mRNA expression in cultured stellate cells [3, 23]. The signaling pathways tbr TGF-[31 involve phosphorylation by activated TGF-13 receptors of cytoplasmic proteins, Smads, which are translocated to the nucleus and interact with other transcriptional factors or coactivators to stimulate pro-collagen gene expression and extracellular matrix deposition [ 1]. Recently, it was reported that hepatic 8-OHdG was immunohistochemically detected in 92% of patients with primary biliary cirrhosis [22]. The tissue 8-OHdG level has been recognized as a useful marker for estimating DNA damage induced by oxidative stress [14, 15, 42]. Increased plasma MDA or lipid peroxide levels have also been reported in cirrhotic patients [6] or patients with obstructive jaundice similar to the present BDL model [50]. These observations suggest that bile duct obstruction in humans and animals induces oxidative stress, as re-
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flected in increased plasma levels of lipid peroxides, with depletion of endogenous antioxidants and oxidative damage to hepatic mitochondrial DNA. Although in the present study we only measured MDA levels as a marker of lipid peroxidation, other markers of lipid peroxidation such as 4-hydroxynonenal (peroxidation product of co-6 potyunsatuarated fatty acids) and F2isoprostanes (peroxidation product of arachidonic acid) have been reported to be increased in several disease states including cirrhosis [14, 32, 33]. It would be interesting to evaluate whether levels of 4-hydmxynonenal or F2isoprostanes are also increased and associated with pathophysiology in BDL rats. In the present study, we also observed several progressively altered parameters such as in plasma MDA, hepatic 8-OHdG, procollagen I and III mRNA expression, and hepatic mitochondrial respiratory control ratio in BDL rats at 2 and 4 weeks after ligation. A previous study of BDL rats at 2, 7, 14 and 21 days showed that there are progressive increases in hepatic mRNA expression of basic fibroblast growth factor, epidermal growth factor, and TGF-131 [34]. In this animal model, progressive changes in the severity of liver fibrosis [24], hepatomegaly [24, 57], portal hypertension [9, 30], and the systemic hyperdynamic state [30] have also been reported. Taken together, these observations suggest a possibility that there might be progressive structural and functional derangements after bite duct ligation in rats up to 4 weeks. In summary, there were mitochondrial dysfunctions and biochemical and molecular biological abnormalities in the liver related to oxidative stress after bile duct ligation. These may contribute to the fibrogenetic processes in the liver.
Acknowledgments This study was supported in part by grants from the National Science Council (NSC 89-2320-B-010-111 and 90-2320-B-010-047), and the National Research Institute of Chinese Medicine (NRICM90104) in Taiwan.
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