Pyrrolidine dithiocarbamate protects against thioacetamide-induced fulminant hepatic failure in rats

Journal of Hepatology 36 (2002) 370–377 www.elsevier.com/locate/jhep

Pyrrolidine dithiocarbamate protects against thioacetamide-induced fulminant hepatic failure in rats Rafael Bruck 1,*, Hussein Aeed 1, Ron Schey 1, Zipora Matas 2, Ram Reifen 3, Gidi Zaiger 3, Ayala Hochman 4, Yona Avni 1 1

Department of Gastroenterology, E. Wolfson Medical Center, Holon 58100, Israel 2 Department of Biochemistry, E. Wolfson Medical Center, Holon 58100, Israel 3 Faculty of Agriculture, Sackler School of Medicine, Rehovot, Israel 4 Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel

Background/Aims: Reactive oxygen species and nuclear factor kappa B (NF-kB) activation have been implicated in the pathogenesis of cell injury in experimental models of liver damage. The aim of the present study was to examine whether pyrrolidine dithiocarbamate (PDTC), an anti oxidant and inhibitor of NF-kB activation, would prevent hepatic damage induced in a rat model of thioacetamide (TAA)-induced liver failure. Methods: Fulminant hepatic failure was induced in the control and treatment groups by two intraperitoneal injections of TAA (either 300 or 400 mg/kg) at 24-h intervals. In the treatment groups, rats were treated also with PDTC (60 mg/kg/24 h, i.p.), initiated 24 h prior to TAA. Results: Liver enzymes, blood ammonia, and hepatic levels of thiobarbituric acid reactive substances (P , 0.001) and protein carbonyls (P , 0.05) were significantly lower in rats treated with PDTC compared to TAA only. Liver histology and the survival rate in the PDTC-treated rats were also improved (P , 0.01 compared to TAA only). NF-kB activation, 2 and 6 h after TAA administration, was inhibited by PDTC. Conclusions: In a rat model of fulminant hepatic failure, the administration of PDTC attenuated liver damage and improved survival. This effect may be due to decreased oxidative stress and inhibition of NF-kB activation. q 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. Keywords: Fulminant hepatic failure; Thioacetamide; Pyrrolidine dithiocarbamate; Nuclear factor kappa B; Reactive oxygen species; Oxidative damage

1. Introduction Nuclear factor kappa B (NF-kB) is a ubiquitous transcription factor involved in the up-regulation of many proinflammatory genes [1,2]. NF-kB binding sites in the promoter region have been demonstrated for a variety of inducible cell adhesion molecules [3], and cytokines, such as tumor necrosis factor (TNF)-a [4], which are implicated in the pathogenesis of organ failure after shock and sepsis. At least five genes belong to the NF-kB family, but most commonly dimers are composed of the Rel A (p65) and NF-kB1 (p50) or NF-kB2 (p52) subunits [1,5]. In most Received 19 January 2001; received in revised form 24 October 2001; accepted 17 November 2001 * Corresponding author. Tel.: 1972-3-502-8499; fax: 1972-3-503-5111. E-mail address: [email protected] (R. Bruck).

cell types, NF-kB dimers are sequestered in an inactive cytoplasmic complex by binding to its inhibitory subunit, IkB. Upon stimulation, IkB undergoes phosphorylation, and this phosphorylation is followed by ubiquitination and rapid degradation by a proteasome-dependent pathway [6–9]. This pathway allows translocation of free, active NF-kB complexes into the nucleus where they bind specific DNA motifs in the promoter/enhancer regions of target genes and activate transcription. Studies with a variety of cultured cell lines suggest that reactive oxygen species could be involved and/or modulate the signal transduction pathway leading to the phosphorylation and ubiquitination of the cytosolic NF-kB complex. This hypothesis is based on several observations: in certain cell lines, hydrogen peroxide was able to directly activate NF-kB, and a variety of antioxidants and antioxidant

0168-8278/02/$20.00 q 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. PII: S01 68- 8278(01)0029 0-2

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enzymes such as catalase, down-regulated TNF-a-mediated NF-kB activation [10–13]. Reactive oxygen species are thought to contribute to the pathogenesis of various liver diseases such as acetaminophen overdose, hemochromatosis, alcoholic liver injury, toxin exposures and viral hepatitis [14–17]. Proinflammatory cytokines, especially TNF-a and several other interleukins are also thought to play a role in cell injury of acute fulminant hepatitis [18,19]. It has been shown in a recent study, that the hydroxyl radical scavenger dimethylsulfoxide (DMSO) was able to inhibit endotoxin-induced NF-kB activation, suppressed TNF-a release, attenuated intercellular adhesion molecule-1 (ICAM-1) mRNA formation and significantly reduced liver injury [20]. In a previous study DMSO was shown to completely prevent fulminant hepatic failure (FHF) in rats induced by the hepatotoxin thioacetamide (TAA). This effect was associated with increased hepatic levels of methanesulfinic acid, which is formed from the interaction of DMSO and hydroxyl radicals [21]. Because NF-kB activation is a common pathway that mediates the upregulation of genes encoding for inflammatory mediators such as adhesion molecules and proinflammatory cytokines, its inhibition may reduce the inflammatory response in FHF. In the present study, we examined the effect of pyrrolidine dithiocarbamate (PDTC), an antioxidant and inhibitor of NF-kB activation [22], in TAA-induced FHF in rats. We demonstrate that the administration of PDTC inhibited TAA-induced NF-kB activation, decreased lipid and protein peroxidation, reduced hepatic necroinflammation and improved survival rate.

2. Materials and methods 2.1. Animals and materials Male Wistar rats (250–300 g), obtained from the Tel-Aviv University animal breeding center, were kept in the animal breeding house of the Wolfson Medical Center and fed a Purina chow ad libitum. Animals were kept in a 12:12-h light/dark cycle at constant temperature and humidity. All rats had free access to tap water during the week before the beginning of the study. Animals received humane care and were treated according to institutional guidelines. TAA and PDTC were purchased from Sigma Chemical Co. (St. Louis, MO).

2.2. Induction of hepatic damage Rats were given intraperitoneal injections of TAA either 300 or 400 mg/ kg, twice at 24-h intervals, as previously described [23,24]. Supportive therapy by subcutaneous administration of 5% dextrose (25 ml/kg) and NaCl 0.9% with potassium (20 meq/l) every 12 h was given to avoid weight loss, hypoglycemia and renal failure [25].

2.3. Experimental design Five groups of rats were studied: (a) TAA 300 mg/kg; (b) TAA 400 mg/ kg; (c) TAA 300 mg/kg 1 PDTC 60 mg/kg/24 h, started 24 h before the first

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TAA injection; )d) TAA 400 mg/kg 1 PDTC 60 mg/kg, as in c; (e) PDTC 60 mg/kg only. Each treatment group consisted of six rats.

2.4. Evaluation of liver injury Serum aminotransferase levels, blood ammonia and liver histology were determined 48 h following the first TAA injection.

2.5. Survival rate The survival rate was determined 52 h after the first TAA injection in separate groups of rats. Three groups of six rats were used for each treatment protocol, and the results for each treatment are given as mean ^ SD of the three groups.

2.6. Preparation of liver homogenate Liver tissue (5 g) was cut into small pieces using a razor blade, and homogenized after dilution in H2O 1:10 w/v. Liver homogenate was centrifuged at 900 £ g for 5 min, and then the supernatant was collected and centrifuged at 9000 £ g in a Sorvall centrifuge for 30 min. Clear supernatant was used for assessment of thiobarbituric acid reactive substances (TBARS) and protein carbonyls.

2.7. Determination of hepatic TBARS TBARS were measured and expressed as nmol/g wet tissue using the thiobarbituric acid method [26]. Briefly, To 1 ml of 10% liver homogenate with 1.15% KCl were added 2 ml of fresh solution 15% w/v TCA, 0.375% w/v TBA, 0.25 ml/l HCl. The mixture was heated at 95 8C for 15 min. The solution was cooled to room temperature using tap water and centrifuged at 300 £ g for 10 min. Absorption of the supernatant was determined spectrophotometrically at 532 nm. The amount of TBARS was expressed in terms of malondialdehyde using 1,1,3,3-tetramethoxypropane as a standard. The level of TBARS was expressed as nmol of malondialdehyde/g wet tissue.

2.8. Determination of protein carbonyls Protein oxidation was measured by assessing protein carbonyls by both enzyme-linked immunosorbent assay (ELISA) [27] and Western blot [28] techniques, based on the recognition of protein-bound compound 2,4-dinitrophenylhydrazine (DNP) by anti-DNP antibodies. For both methods, we used supernatants of crude liver homogenates that were centrifuged at 144 000 £ g for 1 h.

2.9. Determination of NF-k B activation 2.9.1. Isolation of nuclear proteins Nuclear proteins from liver tissue were isolated by the modified method of Dignam et al. [29]. Briefly, tissue samples were homogenized with an Ultra-Turrax homogenizer (IKA, Staufen, Germany) in 3 ml ice-cold buffer A (10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 1 mM dithiothreitol (DTT), 0.2 mM phenylmethylsulfonyl fluoride (PMSF) (Sigma)). Homogenates were incubated for 10 min on ice and centrifuged at 25 000 £ g for 15 min at 4 8C (RC5C Sorvall Instruments, Du Pont, Wilmington, DE). The crude nuclear pellet was rinsed once with buffer A, centrifuged at 10 000 £ g for 15 min at 4 8C, and then was resuspended in 200 ml of buffer B (20 mM HEPES (pH 7.9), 25% glycerol (v/v), 1.5 mM MgCl2, 0.2 mM ethylenediaminetetraacetic acid (EDTA), 0.2 mM PMSF, 1 mM DTT, 1.2 M KCl), and incubated on ice for 30 min. Nuclear proteins were recovered after centrifugation at 25 000 £ g for 30 min at 4 8C, and proteins were stored at 270 8C. Protein concentrations were determined with the Protein Assay Reagent kit of Bio-Rad (Bio-Rad Laboratories, Richmond, CA).

2.9.2. Electrophoresis mobility shift assay Double standard NF-kB consensus oligonucleotide probe (5 0 -AGT TGA

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GGG GAC TTT CCC AGGC-3 0 ) was purchased from Promega Corp., Madison, WI and end labeled with [g- 32P]adenosine triphosphate (3000 mCi/mmol, Amersham, Arlington Heights, IL). Binding reactions, containing 35 pmol (1.75 pmol/ml) of oligonucleotide and 1 mg of nuclear protein were conducted at room temperature for 20 min in a total volume of 10 ml in binding buffer (10 mM Tris–HCl (pH 7.5), 50 mM NaCl, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 4% glycerol (v/v), and 0.5 mg poly(dI:dC)). For competition reactions, unlabeled oligonucleotide was added 5 min prior to addition of radiolabeled probe. Following the binding reactions, gel loading buffer was added and the reaction subjected to nondenaturing 4% polyacrylamide gel electrophoresis in 0:25£ TBE buffer (45 mM Tris-borate, 1 mM EDTA, pH 8.0) at 100 V/20 mA for 2 h. Gels were vacuum dried and exposed to X-ray film (Kodak, Biomax MS) at 270 8C.

2.9.3. Liver histopathology For liver histopathology analysis, midsections of the left lobes of the liver were processed for light microscopy. This processing consisted of fixing the specimen in a 5% neutral formol solution, embedding the specimens in paraffin, slicing sections 5 mm thick, and staining the sections with hematoxilin and eosin (H&E). The tissue slices were scanned and scored blindly by two expert pathologists. The degree of inflammation and necrosis were expressed as the mean of 10 different fields within each slide that had been classified on a scale of 0–3 (normal, 0; mild, 1; moderate, 2; severe, 3).

2.10. Statistical analysis The data are presented as the mean ^ SD. The significance of differences between different groups was determined by Student’s t-test.

Fig. 1. Blood ammonia in TAA-induced fulminant liver failure. Blood ammonia was significantly lower in the groups of rats treated with TAA 1 PDTC compared to TAA only (300 or 400 mg/kg). Mean ^ SD, n ¼ 6 rats in each treatment group; **P , 0:001 compared to TAA only.

the rats which received TAA 300 mg/kg was elevated 5fold compared to the rats that were treated also with PDTC (13.6 ^ 1.3 vs. 2.8 ^ 0.7 mg/ml, P , 0:0001, Fig. 1). A similar protective effect of PDTC was observed in the rats that received TAA 400 mg/kg (39.3 ^ 5.8 vs. 7.9 ^ 2.1 mg/ ml, P , 0:0001).

3. Results 3.1. Effect of TAA on liver enzymes and blood ammonia Severe liver injury, manifested by elevation of serum aminotransferase levels and blood ammonia was observed 48 h after the administration of 300 or 400 mg/kg TAA (Table 1 and Fig. 1).

3.3. Hepatic levels of TBARS In the TAA 1 PDTC-treated rats, hepatic TBARS were significantly lower compared to the TAA-treated animals (9.7 ^ 0.9 vs. 14.0 ^ 2.1/g wet tissue, P ¼ 0:006, Fig. 2A). 3.4. Hepatic levels of protein carbonyls

3.2. Inhibition of TAA-induced liver injury by PDTC In the rats treated with TAA and PDTC the levels of ALT and AST, measured 48 h after the first TAA injection were significantly decreased, compared to the animals which received TAA only (Table 1). Blood ammonia levels in

The hepatic levels of protein carbonyls, measured by ELISA (Fig. 2B) and Western blot analysis (Fig. 3) were elevated after TAA administration and significantly decreased in the livers of the rats which received PDTC in addition to TAA.

Table 1 Serum levels of liver enzymes in TAA-induced fulminant hepatic failure a

3.5. Liver histopathology

Compounds used

AST (IU/l)

ALT (IU/l)

PDTC only TAA (300 mg/kg) TAA (300 1 PDTC) TAA (400 mg/kg) TAA (400 1 PDTC)

163 ^ 26 2590 ^ 400 762 ^ 386** 7847 ^ 640 2600 ^ 1048**

61 ^ 10 940 ^ 269 302 ^ 167** 1367 ^ 215 434 ^ 195**

a Hepatic enzymes determined after two i.p. injections of TAA either 300 or 400 mg/kg at 24-h intervals, 48 h after the first administration. TAA, thioacetamide; PDTC, pyrrolidine dithiocarbamate, 60 mg/kg i.p. Values are mean ^ SD, n ¼ 6 in each group. **P , 0:001 compared to TAA alone.

Histopathologic examination of liver specimens showed severe hepatic necrosis and inflammation in the livers of the rats treated with TAA only (300 or 400 mg/kg, Fig. 4A,B, respectively). In contrast, no evidence of necrosis and a very mild degree of pericentral inflammation were observed in the livers of the TAA 1 PDTC-treated rats (Fig. 4C,D and Table 2, P , 0:001). Hepatic necrosis and inflammation were observed mostly around the central vein, and less in the hepatic lobule and the periportal area. Liver histopathology in control rats treated only with PDTC (60 mg/kg at 24h intervals) was normal (not shown).

R. Bruck et al. / Journal of Hepatology 36 (2002) 370–377

Fig. 2. (A) Hepatic levels of TBARS in the livers of rats treated with TAA 1 PDTC were significantly lower compared to TAA only. Mean ^ SD, n ¼ 6 rats; *P , 0:01. (B) Hepatic levels of protein carbonyls were lower in the PDTC-treated rats. Measurement by ELISA, performed in triplicate. Mean ^ SD, n ¼ 6 rats; **P , 0:001. TAA, 400 mg/kg; PDTC, 60 mg/kg.

3.6. TAA-induced NF-k B activation is inhibited by PTDC To study the effect of TAA administration on NF-kB binding activity, electrophoresis mobility shift assays were performed. Although no specific NF-kB binding activity was detected in the unstimulated state (not shown), TAA administration induced NF-kB binding activity as determined after 2 and 6 h, which was significantly attenuated by PDTC treatment (Fig. 5). 3.7. Survival rate

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rate. At the molecular level, TAA administration resulted in oxidative stress manifested as increased hepatic levels of lipid peroxides and oxidized proteins, and activation of NFkB. Administration of PDTC attenuated hepatic damage, as shown by the lower levels of liver enzymes and blood ammonia, the marked improvement in liver histology, and the reduced levels of TBARS and protein carbonyls. TAA is a thiono-sulfur-containing compound, which has liver-damaging and carcinogenic effects. Shortly after administration, it undergoes extensive metabolism by the mixed function oxidase system to acetamide which is devoid of liver necrotizing properties, and thioacetamideS-oxide [30]. Thioacetamide-S-oxide is metabolized by cytochrome P-450 monooxygenases to further compounds, including the very reactive compound thioacetamide-Sdioxide [31,32]. The binding of this metabolite to tissue macromolecules may be responsible for the production of hepatic necrosis, induction of apoptosis [33], perturbation of mitochondrial activity [34,35], and elevation of serum cytokine levels [31]. Numerous studies in rats and cultured cells indicated the involvement of oxidative stress in the etiology of TAA-induced liver damage. In these studies, TAA caused lipid peroxidation [21,36–39], increased susceptibility of hepatocytes to in vitro lipid peroxidation [40], decreased GSH/GSSG ratio [38,40–42], increased GSH synthesis [36], alterations in low molecular weight [33] and enzymatic antioxidant [41–43]. A protective effect was demonstrated by antioxidants such as DMSO [21] and Nacetylcysteine [37], aminoguanidine, [38], prostaglandin E2 [39] and the calcium channel blocker verapamil [40]. However, several studies could not fully confirm this oxidative stress hypothesis [21,44–46]. The mechanism of reactive oxygen species (ROS) production implicated the enzyme xanthine oxidase [47], and the cytokines TNF-a and interleukin (IL)-1 [37]. In accordance with previous studies, we demonstrated in this work that treatment of rats with TAA caused oxidative stress to the liver, as shown by the enhanced lipid peroxidation, assayed as TBARS, and increased protein carbonyls. NF-kB is a ubiquitous transcription factor that plays a pivotal role in cell death and survival pathways [1–6]. It can be activated by a variety of physiological and nonphy-

The survival rate 52 h after the first TAA injection was significantly higher in the groups that were treated with TAA 1 PDTC compared to the rats treated with TAA only (P ¼ 0:004 for TAA 300 mg/kg and P ¼ 0:002 for TAA 400 mg/kg, Fig. 6). 4. Discussion In the present study, we have shown that treatment of rats with TAA induced severe liver damage associated with elevated levels of liver enzymes and blood ammonia, as well as hepatic necroinflammation, and a high mortality

Fig. 3. Measurement of hepatic levels of protein carbonyls in crude liver extract by Western blot analysis. A representative gel of three samples. TAA, 400 mg/kg; PDTC, 60 mg/kg.

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Fig. 4. Liver histology of rats with fulminant hepatic failure induced in all groups by two consecutive injections of TAA at 24-h intervals. Rats were killed and livers fixed 48 h after the first TAA injection. (A) TAA 300 mg/kg; (B) TAA 400 mg/kg. Both sections show severe liver necrosis and inflammation mostly around the central veins. (C) TAA 300 mg/kg 1 PDTC 60 mg/kg; (D) TAA 400 mg/kg 1 PDTC 60 mg/kg. Only minimal hepatic necrosis and inflammation is present in the pericentral area. H&E, £80.

siological stimulating signals relevant to pathophysiology, including proinflammatory cytokines such as TNF or IL-1, viral infection or expression of certain viral gene products, B- or T-cell activation, ROS and UV irradiation. Activation Table 2 Liver histology in TAA-induced fulminant hepatic failure a Treatment

Inflammation pericentral (0–3)

Necrosis pericentral (0–3)

PDTC only TAA (300 mg/kg) TAA 300 1 PDTC TAA (400 mg/kg) TAA 400 1 PDTC

0 2.0 ^ 0.0 0.2 ^ 0.4** 2.5 ^ 0.5** 1.3 ^ 0.5*

0 2.0 ^ 0.0 0.2 ^ 0.5** 2.5 ^ 0.5** 1.2 ^ 0.5*

a Rats were killed and livers fixed 48 h after the first TAA injection. TAA, thioacetamide; PDTC, pyrrolidine dithiocarbamate (60 mg/kg). Liver damage in response to TAA was observed mostly in the pericentral areas, whereas the periportal zones were relatively intact. Values are mean ^ SD, n ¼ 6. **P , 0:0001, *P , 0:01 compared to TAA alone.

of NF-kB regulates the expression of many genes involved in inflammatory, immune, and apoptotic responses. Many antioxidative agents can suppress NF-kB activation, including N-acetylcysteine, vitamin E, dithiocarbamates and heavy metal chelators [48], while several NF-kB activators induce oxidative stress as well as other more specific effects [48]. We have shown in this study that treatment of rats with TAA caused activation of NF-kB. In another study that links TAA with NF-kB activation [36], TAA increased binding of NF-kB, the redox-sensitive activator protein (AP)-1 and the antioxidant response element (ARE) [36]. At present, it is too early to infer about the mechanism by which TAA caused liver damage. It may be a direct effect, either by giving rise to ROS or activation of NF-kB, or by an indirect response in which ROS generated by TAA activate NF-kB. In a rat model of CCl4-induced liver necrosis, liver injury was caused by oxidative stress [14,15]. In this model, ROS induced NF-kB activation and the expression of a number of

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Fig. 5. Electrophoresis mobility shift assay showing increased NF-kB binding activity in the nucleus, 2 and 6 h after the administration of TAA (400 mg/kg). This activation was attenuated by PDTC (60 mg/kg).

cytotoxic factors, including TNFa [49], indicating that NFkB activation may link oxidative stress, cytotoxic cytokines, and liver cell necrosis [49]. Pyrrolidine dithiocarbamate (PDTC) functions as an antioxidant due to two structural features: direct scavenging of ROS by the dithiocarboxy group, and chelating activity for heavy metal ions that may catalyze formation of ROS. In addition, PDTC protects against NF-kB-mediated pathological effects induced by various stimuli including LPS and cytokines [50–53]. Furthermore, it has been shown that PDTC has a concentration-dependent effect on NF-kB activation, causing inhibition at low but not high concentrations [22,54]. This might be explained by recent observations, which suggested that the ability of PDTC to inhibit NFkB activation was associated with its ability to increase intracellular Zn 21 levels, by promoting Zn 21 influx only at low concentrations [54,55]. In endothelial cells, elevation of the intracellular Zn 21 level by pyrithione, a zinc ionophore, inhibited NF-kB activation [56]. Thus, PDTC may act through an additional mechanism, unrelated to its antioxidative or metal chelating activity. In our study, PDTC attenuated the effects of TAA: it decreased liver damage, and inhibited oxidative stress and NF-kB activation. To the best of our knowledge, this is the first report on the protection by PDTC against the deleterious cellular effects of TAA. The exact mechanism by which PDTC inhibits NF-kB activation is still unclear. Studies in intact cells have shown that PDTC inhibits NF-kB activation by all inducers (i.e. phorbol esters, IL-1, lipopolysaccharide and TNF-a) with low toxicity [22], but had no inhibitory effect on the activation of

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other transcription factors such as AP-1, Sp-1, and CREB [57]. Because non-thiol metal chelators can also inhibit NFkB activation, the inhibitory effect of PDTC on NF-kB may rely on both its properties. This could explain why dithiocarbamates are much more potent inhibitors of NF-kB activation than sulfhydryl compounds, which are not strong chelators of metal ions [22]. It has been suggested that the effect of PDTC is by suppression of a reaction required for release of IkB from NF-kB in the cytosol. Because PDTC could block this reaction in response to every NF-kB inducer tested [10,22], it seems to interrupt with the activity of a common messenger, produced by different signaling pathways which might be ROS, probably hydroxyl radicals [22]. In previous studies, we have shown that the TNFa antagonists pentoxifylline and soluble TNF receptor, had no protective effect on TAA-induced liver damage [21], suggesting that TNFa did not play a major role in mediating liver damage in this model. However, the inhibition of NFkB activation by PDTC may still attenuate hepatic damage by inhibiting the production of other proinflammatory cytokines which are upregulated by NF-kB activation (e.g. IL-2, -6, and -8), and by blocking ICAM-1 mRNA formation and neutrophil accumulation [13,20]. In summary, the findings described in the present work suggest a role for ROS-induced NF-kB activation in the pathogenesis of TAA-induced liver damage, and indicate that blockade of one or both pathways by PDTC has a potent anti-inflammatory effect. Additional studies are required to fully elucidate the exact mechanism and sequence of events by which TAA induces liver damage and the protective effect(s) of PDTC. These studies will reveal the therapeutic potential of PDTC in decreasing hepatic necrosis and inflammation.

Fig. 6. Survival rate in TAA-induced hepatic damage (300 or 400 mg/ kg), with and without PDTC. The survival rate was determined 52 h after the first TAA injection in separate groups of rats. For each treatment protocol, three groups of six rats each were used, and the percentage of survivors for each treatment protocol is given as mean ^ SD of three separate experiments. *P , 0:01.

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