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Annals of Clinical & Laboratory Science 31:383-390 (2001)
© 2001 Association of Clinical Scientists

Free Radical-Triggered Hepatic Injury of Experimental Obstructive Jaundice of Rats Involves Overproduction of Proinflammatory Cytokines and Enhanced Activation of Nuclear Factor {kappa}B

Tsan-Zon Liu1,2, King-Teh Lee3, Chi-Liang Chern1,4, Jiin-Tsuey Cheng4, Arnold Stern5 and Li-Yu Tsai6
1 Department of Medical Research, Yuan’s General Hospital, Kaohsiung, Taiwan
2 Department of Medical Technology, Fooyin Institute of Technology, Ta-Liao, Kaohsiung Hsien, Taiwan
3 Department of Surgery, Kaohsiung Medical University, Kaohsiung, Taiwan
4 Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan
5 Department of Pharmacology, New York University Medical Center, New York City, New York, USA
6 Department of Clinical Biochemistry, Kaohsiung Medical University, Kaohsiung, Taiwan.

Address correspondence to Li-Yu Tsai, Ph.D., Department of Clinical Biochemistry, School of Technology for Medical Sciences, Kaohsiung Medical University, Taiwan 807; tel 886 7 312 1101, x7052; fax 886 7 237 0544; e-mail tsliyu{at}cc.kmu.edu.tw.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Excessive production of hydroxyl radicals in blood and liver has previously been demonstrated by us in rats with obstructive jaundice induced by common bile duct ligation (CBDL). In this study, we demonstrate overproduction of superoxide radicals in circulating blood of CBDL rats by the lucigenin-amplified chemiluminescence technique. To pinpoint the molecular agents that mediate these processes, we measured circulating proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-{alpha}), interleukin-1ß ( IL-1ß), and interleukin-6 (IL-6) in controls and CBDL rats. Concentrations of these cytokines in blood of CBDL rats were markedly elevated when compared to the controlsSTNF-{alpha}: 36.7±5.0 vs 13.8±0.5 pg/mL; IL-6: 2,814±1,740 vs 0 pg/mL; IL-1ß: 11.9±2.6 vs 0 pg/mL). The overproduction of free radicals triggered by elevated cytokines in CBDL rats was correlated with the activation of NF-{kappa}B in hepatic tissue. Using the TdT-mediated dUTP nick-end label staining technique, we showed that hepatic tissue sections from CBDL rats had an increase in the apoptotic index (AI). Based on these findings, we propose that the severe hepatic injury in CBDL rats is mediated by a cycle that involves the activation of NF-{kappa}B by combined action of proinflammatory cytokines and reactive oxygen species (ROS). NF-{kappa}B, in turn, initiates the transcription of cytokine genes (eg, IL-6, IL-8, TNF-{alpha}), which triggers hepatic injury, at least in part, by a free radical-mediated apoptotic mechanism. Elevated ROS may be as a positive feedback signal that triggers NF-{kappa}B reactivation; the severe hepatic injury of CBDL rats may result from perpetuation of this vicious cycle.

(received 28 April 2001; accepted 7 June 2001)

Keywords: common bile duct ligation, oxidant stress, cytokines, nuclear transcription factor NF-{kappa}B


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Obstructive jaundice can be induced in the rat by common bile duct ligation (CBDL), which causes parenchymal cell damage that may ultimately lead to liver cirrhosis and portal hypertension [1]. Previously, we demonstrated that the tissue injury induced by obstructive jaundice involves lipid peroxidation [2,3]. Most CBDL animals have been shown to be deficient in fat-soluble vitamins, such as vitamins A and E [4,5]. Because these vitamins are capable of ameliorating secondary tissue damage induced by lipid peroxidation, enhanced oxidative stress could possibly exacerbate secondary tissue damage. Moreover, obstructive jaundice could alter the activities of antioxidant enzymes resulting in the increased production of superoxide (O2•) and hydrogen peroxide (H2O2) [6]. As a result, hydroxyl radical (•OH) can be expected to form through the interplay between O2•, H2O2, and iron via the Haber-Weiss (1) or Fenton (2) reactions (2) [7,8]:


(1)

(2)

We have shown that such reactions occur, as evidenced by increases of multi-hydroxylated salicylate compounds, eg, 2,3-dihydroxybenzoate (2, 3-DHB), 2,5-dihydroxybenzoate (2,5-DHB), in the plasma of CBDL rats after aspirin was given by gavage. To prove that •OH is generated in the liver after CBDL, male rats were treated with ip injection of DMSO prior to CBDL, followed by spectroscopic measurement of methane sulfinic acid (MSA), a stable non-radical compound formed by reaction of •OH with DMSO. The accumulation of MSA was greatly increased in hepatic tissue after CBDL [9]. Even though liver damage observed in the CBDL animal model may be due to the combined effects of overproduction of reactive oxygen species (ROS) and suppressed antioxidative reserve (vitamin A and E deficiencies), the key elements in mediating the oxidative stress caused by CBDL remain elusive.

The objectives of the present study were (a) to investigate whether or not the CBDL model elicits increased release of proinflammatory cytokines, and (b) to see what "mediator" regulates the release of proinflammatory cytokines. Our data suggest that the sustained hepatic injury in CBDL rats involves a noxious cycle of interplay between overproduction of circulating proinflammatory cytokines, oxidant stress (O2•, H2O2, and •OH), and activation of nuclear transcription factor NF-{kappa}B. The NF-{kappa}B activation appears to mediate the regeneration of ROS and proinflammatory cytokines.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Animals.  The protocol was approved by the animal protection committee of Kaohsiung Medical University. Wistar rats (n = 45, male, 12 wk-old) were allowed ad libitum access to standard rat chow and water and were housed in the animal quarters for 3–4 wk. Surgery was performed under pentobarbital anesthesia (60 mg/kg, ip). The rats were randomly assigned to two groups. The control group (Group I) (n = 30, body wt 385 ± 22 g) was sham-operated. The other group (Group II) (n = 15, body wt 332 ± 28 g) underwent bile duct ligation. Blood samples were collected at 14 da after the sham-operation or bile duct ligation for assays of superoxide and cytokines. Samples of hepatic tissue for measurements of apoptotic index were obtained at 4 da after the sham-operation (n = 30 rats) or bile duct ligation (n = 15 rats).

Superoxide assay.  Superoxide was measured by chemiluminescence as described previously [10]. Briefly, 0.2 ml of whole blood was mixed with 0.1 ml of phosphate buffered saline (PBS) buffer, pH 7.4, in a reaction cuvet (model # TLU-21 Tohoku Electronic Co., Sendai, Japan), that comprised a stainless steel cell with magnetic stirrer in an absolutely dark chamber of the chemiluminescence Analyzing System (Tohoku Electronic Co.). After 200 sec, 1.0 ml of lucigenin (Sigma Co., USA, 1 µM in PBS) was injected into the stainless steel cell and photon emission was counted at 10 sec intervals (37°C, atmospheric conditions) for 1000 sec. Total chemiluminescence was calculated by integrating the area under the curve and subtracting the background level (ie, the dark average).

ELISA for proinflammatory cytokines.  Serum samples from the CBDL rats and control rats (sham-operated) at 14 da after surgery were analyzed for IL-6, IL-1ß, TNF-{alpha}, and IFN-{gamma}. These cytokines were measured by ELISA kits (The Biosource International CytoscreenTM, Camarillo, CA, USA) according to instructions in the package insert.

Apoptotic index measured by the TdT-mediated dUTP nick end labeling technique.  The TUNEL stain (TdT-mediated dUTP nick end labeling) (Boehringer-Mannheim, GmbH, Ingleheim, Germany) was performed using 5 µm formalin-fixed, paraffin-embedded sections of liver tissue. The slides were treated with 0.3% H2O2 in methanol for blocking endogenous peroxidase activity for 10 min at 37°C. After washing with phosphate-buffered saline (PBS), pH 7.4, the slides were incubated with proteinase K (10 µg/ml in Tris-HCl buffer, pH 7.6) for 30 min at room temperature. The slides were then washed with PBS and incubated with "Label Solution–TUNEL Reaction" (Boehringer-Mannheim) (1 hr, 37°C). The slides were washed with PBS and then incubated with "Converted POD Solution" (Boehringer-Mannheim) (30 min, 37°C). Visualization was achieved by incubation with diaminobenzidine (Sigma Co., St Louis, MI, USA) and counter-staining with Mayer’s hematoxylin. Dehydration and clearing of the slides were performed in graded alcohol and xylene, respectively. The slides were mounted with Entellan (Merck, Darmstadt, Germany). The labeling index (%) was assayed by light microscopy at 400-x magnification. Results were recorded +/-, +, or ++, which represent < 5%, 5–10%, and >10% apoptotic index, respectively. Positive controls were produced by treating slides with DNAse I (1 mg/ml) for 10 min at room temperature before incubation with the TUNEL reaction mixture. Negative controls were produced by incubating slides with the Label Solution without terminal transferase.

NF-{kappa}B electrophoretic mobility shift assay.  Assays of NF-{kappa}B activity used the procedure of Mihm et al [11]. Briefly, nuclear protein extracts (20 µg) from hepatic tissues of CBDL and control rats were preincubated for 10 min at room temperature in a gel shift binding buffer (20% glycerol, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl, 50 mM Tris-HCl (pH 7.5), and 0.25 mg/ml poly(dl-dc) (Boehringer-Mannheim) for 15 min. After adding 32P-end-labeled NF-{kappa}B binding oligonucleotide (5'-AGT TGA GGG GAC TTT CCC AGGC-3'), as described by Read et al [12], and 3 mM guanosine triphosphate (GTP; Sigma Co.), the mixture was incubated for 20 min at room temperature and loaded on 5% polyacrylamide gel (acrylamide:bisacrylamide 19:1, v:v; 0.045 M Tris, 0.045 M boric acid, 1 mM EDTA). After electrophoresis (1 hr, 12 v/cm, 0.089 M Tris, 0.089 M boric acid, 2 mM EDTA), the gels were dried and exposed overnight to Kodak XAR film with an intensifying screen. NF-{kappa}B expression was confirmed by supershift assay with anti-P65 antibody.

Statistics.  The data were expressed as mean ± SEM. Differences between the groups were considered significant at p <0.05. Statistical analyses were done by one-way ANOVA and Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
Increased production of superoxide radicals in the blood of the CBDL rats.  Lucigenin-amplified chemiluminescence (CL) was used to compare the superoxide radical production in blood samples from control and CBDL rats at 14 da after surgery. As illustrated by the typical results in Fig. 1Go, the total CL in blood of a control rat was 5,469 units, while the total CL in blood of a paired CDBL rat was 60,735 units, which represents ~11-fold increment of O2• production, versus the control.



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  		Fig. 1. Ultra-weak lucigenin-amplified chemiluminescence monitoring of superoxide production in blood samples from sham-operated control and CBDL rats at 4 da after surgery.

 
Liver of CBDL rats has increased apoptotic index.  The TUNEL (TdT-mediated dUTP nick-end labeling) staining technique was used to test if free radical-triggered injury of hepatic tissue in CBDL rats is preceded by apoptosis. As shown by the typical results in Fig. 2Go, formalin-fixed, paraffin-embedded hepatic tissue from a sham-operated control rat exhibited no fluorescent-labeled dUTP incorporation, indicating that hepatocyte DNA was intact intact. In contrast, formalin-fixed, paraffin-embedded hepatic tissue of a paired CBDL rat had extensive fluorescent-labeled dUTP incorporation into hepatocyte DNA, with an estimated apoptotic index of 25%. These findings indicate that the hepatic damage caused by obstructive jaundice in CBDL rats was due, at least in part, to the induction of apoptosis triggered by excessive production of free radicals.



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  		Fig. 2. TUNEL stain labeling technique for estimating apoptotic index (%) for liver sections from sham-operated control and CBDL rats at 4 da after surgery (400x magnification). A: control; B: CBDL.

 
Increased proinflammatory cytokine concentrations in serum of CBDL rats.  As listed in Table 1Go, IL-6 and IL-1ß concentrations were undetectable in serum of control rats at 2 da after sham operation. In contrast, these cytokine concentrations were both markedly elevated in serum of the CBDL rats (IL-6: 2814±1740 pg/ml; IL-1ß:11.9±2.6 pg/ml). In CBDL rats, serum TNF-{alpha} concentration averaged ~3x that of the controls (36.7±5.7 vs 13.8±0.5 pg/ml) and serum IFN-{gamma} concentration averaged ~10x that of controls (48.2±18.2 vs 4.5±0.4 pg/ml).


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  		Table 1. Concentrations of proinflammatory cytokines in blood serum samples from sham-operated control and CBDL rats at 14 da after surgery. The results are expressed as mean ± SEM; ND = not detected.
 
NF-{kappa}B regulates the release of proinflammatory cytokines and increased production of free radicals in CBDL rats.  Hepatic tissues from the sham-operated and the CBDL rats were compared for the propensity of NF-{kappa}B expression. As shown in Fig. 3Go, hepatic NF-{kappa}B expression in CBDL rats was much more pronounced than in the sham-operated control, based on an electrophoretic mobility shift assay (EMSA) and the supershift technique using anti-P65 antibody. These observations were attended by abrupt increases of proinflammatory cytokines in serum specimens of CBDL rats. The observed increase of free radical production may be be caused by a sudden increase of proinflammatory cytokine production.



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  		Fig. 3. Overexpression of NF-{kappa}B in nuclear protein extracts from livers of 2 CBDL rats, compared to a sham-operated control (C). NF-{kappa}B expression was assessed by the electrophoretic mobility shift assay and confirmed by the supershift technique using anti-P65 antibody.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
As previously reported, the damage of hepatic tissue in CBDL rats probably occurs by a mechanism involving necrosis, with mitochondrial swelling and distorted cristae [9]. We now observe a pronounced increase of apoptotic index (~25%), as detected by the TUNEL technique in formalin-fixed, paraffin-embedded sections of liver from CBDL rats. This indicates that, in addition to necrosis, the hepatocytes undergo apoptosis.

Regarding the mechanism(s) of hepatic injury, we speculate that oxidative stress mediated by reactive oxygen species (ROS) may be one of the causative factors. We previously presented evidence of accelerated generation of hydroxyl radicals (•OH) in plasma and liver of CBDL rats [9]. In addition, CBDL rats were deficient in antioxidant enzymes, resulting in increased production of O2• and H2O2 [6]. In the present study, overproduction of O2• in the blood of CBDL rats was confirmed by lucigenin-amplified chemiluminescence. Since it is well-recognized that various forms of organ damage involve ROS-mediated oxidative stress, our data suggest that accelerated generation of ROS (O2•, •OH, and H2O2) may, at least in part, have an important role in the pathogenesis of hepatic injury associated with obstructive jaundice.

Endotoxemia can occur in jaundiced patients and animals with experimental biliary obstruction [13–15]. Endotoxemia is known to induce release of proinflammatory cytokines, such as TNF-{alpha} and IL-6 [15]. Experimental evidence has indicated that circulating cytokines are capable of eliciting the overproduction of ROS. For example, TNF-{alpha} was shown to induce oxidative stress by overproduction of O2•, H2O2, and •OH [16–19]. In this study, the concentrations of circulating proinflammatory cytokines, such as TNF-{alpha}, IL-6, and IL-1ß, increased dramatically in CBDL rats. This observation helps to link the overproduction of ROS, proinflammatory cytokines, and hepatic injury.

Gene expression in cells is governed by nuclear transcription factors. NF-{kappa}B is a ubiquitous transcription factor that controls the inducible expression of a variety of genes. NF-{kappa}B is composed of two DNA-binding subunits, P50 and P65 [20,21]. In the unstimulated condition, P50/P65 heterodimer is complexed with I{kappa}B, an inhibitory subunit that prevents the migration of P50/P65 heterodimer to the nucleus [22].

Many agents can induce the DNA-binding activity of NF-{kappa}B. Among these are lipopolysaccharide (LPSs), viruses, inflammatory cytokines (TNF-{alpha}, IL-1ß), and UV irradiation [23–27]. Recently, Schmidt et al [28] showed that, in mouse JB6 cells, overexpression of Cu/Zn-SOD increases NF-{kappa}B activation by TNF-{alpha}. In contrast, over-xpression of catalases decreases NF-{kappa}B activation. These data imply an essential role for ROS, probably H2O2, in NF-{kappa}B activation.

We observed pronounced activation of NF-{kappa}B in liver of CBDL rats. NF-{kappa}B may regulate the abrupt release of proinflammatory cytokines, such as TNF-{alpha}, IL-6 and IL-1ß, by initiating the transcription of their genes in CBDL rats [21]. These cytokines may then play a pivotal role in initiating the overproduction of ROS [12,16–19]. As to the possible mechanism(s) for activation of NF-{kappa}B in CBDL rats, overproduction of ROS is implicated for the following reasons: First, most inducers of NF-{kappa}B seem to rely on the production of ROS, as demonstrated by the inhibitory effect of antioxidants (eg, N-acetyl-cysteine) and the activation by H2O2 [27,29–30]. Second, it is speculated that ROS can directly activate NF-{kappa}B by degrading or modifying I{kappa}B in the cytoplasmic P50/P65/I{kappa}B complex.

Based on these considerations, excessive production of ROS in CBDL rats may enhance the activation propensity of NF-{kappa}B. Studies by others have shown that TNF-{alpha} is capable of inducing MnSOD mRNA [31]. The MnSOD gene contains in its upstream region putative NF-{kappa}B binding sites, which may be involved in transcriptional up-regulation of the MnSOD gene by oxidants. Ultimately, over-expression of MnSOD in mitochondria dismutates O2• into H2O2 which, if overproduced beyond the detoxification capacity of hepatic catalase, will be a signal for reactivation of NF-{kappa}B.

Alternatively, the binding of NF-{kappa}B to DNA promoter sequences may be dependent on the intracellular reducing condition [32]. Glutathione (GSH) plays a primary role in maintaining the intracellular reducing environment and GSSG has been proposed as being necessary to initiate the activation of NF-{kappa}B, while GSH is required for optimal NF-{kappa}B binding [33]. In this regard, owing to the deficiency of fat-soluble vitamins (eg, vitamins A and E) and excessive elevation of intracellular ROS in CBDL rats, the endogenous GSH reserve can be depleted, resulting in increased generation of GSSG. This situation may create a favorable condition for NF-{kappa}B activation.

In conclusion, it appears that over-production of ROS by CBDL rats has a direct signaling role for the activation of NF-{kappa}B. Activated NF-{kappa}B, in turn, promotes the expression of proinflammatory cytokine genes and induces transcription of their encoded cytokines (eg, TNF-{alpha}, IL-6, IL-1ß). These cytokines can induce oxidative stress by promoting overproduction of ROS. Thus, oxidant stress may perpetuate liver damage by inducing cytokine gene expression via activation of NF-{kappa}B while exerting direct injurious effects on cells (Fig. 4Go). The mechanisms outlined in this cycle may furnish the basis for therapeutic approaches to ameliorating the hepatic injury caused by obstructive jaundice.



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  		Fig. 4. Model of a mechanistic cycle that relates the key factors involved in the hepatic injury of CBDL rats.

 


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
The authors thank Ms. Mana Chi for expert assistance in typing this manuscript.

This study was supported in part by grants from the National Science Council-ROC (NSC 88-2314-B037-060) and National Institute of Environmental Health Sciences-USA (ES-03425, to A.S.).


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

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