reatment with PGE2RA. Results: 1. Apoptusis indexes 6 hours after treatment with CHll alone CHll + indomethacin and Cttl 1 + PGE21Lr were 32%, 43% and 16%, respecuvely. 2 Both the Bcl-xL and Mcl- 1 protein expression levels significantly increased 30-180 minutes atler treatment vath PGE2RA, while indomethacin did not change the expression levels of hese proteins, Conclusion: Direct induction of anti-apoptotic Bcb2 family proteins plays an important role in the hepatocyte-protecuve effects nidnced by" PGE2RA,

$878

kycopene Attenuates Alcohol-Induced Apoptosis and Oxidative Stress in HepG2 Cells Overexpressing CYP2E1 Youqing Xu Maria A Leo, Charles S Lieber

Ethanol protnotes hepatic apoptosis but the mechanism is still debated To test the hypothesis hat this may be secondary to the oxidative stress generated by Wtochmme P4502E1 (CfP2E 1) we tested the effects of the antioxidant lycopene on oxidative stress and apoptosis m HepG2 cells ovelvxpressmg CYP2E1 To that effect, a ftdbtength human CYP2E1 cDNA (kindly provided by Dr F h Gonzalez, National Cancer Institute, Bethesda, MD) was inserted into t}-~e EcoRl restriction s~te of the pCl-neo expression vector A HepG2 subclone (2El) stably ave,expressing CYP2E I and a control subclone (Neo) were established. Northern and Westenr blots showed a highly expressed CYP2E1 The 2E1 cells had an active cyto- chrome CYP2E1 confirmed by the conversion of p-nitrophenol to 4-nitrocatechol (053_+010 mnolgmin/mg protein) The ceils were exposed tar 5 days to lOOmM ethanol and 10}~M lycopene or an equal w9lume of placeba~ (vehicle) kindly donated by BASF (ludwigshakn, Germany) ghe uptake of lycopene by the 2El and by the Neo cells was similar (801 • and 737• respectively) Compared to the Neo cells, ethanol signiiicantly increased apoptosis in 2E1 cells: 13.30 • 0.57 vs. 10.25 • 0.75%; p<0.05 (by [low cytometry') and 15• 1.06 vs 7.67_+076%; p<0 001 (by" TUNEL assay'). This effect was attenuated by/ycopene in 2El cells (7.67 _+ 0.26 by flow cytometry and 4 16 • 0.48% by TUNEL assay p<0 001). This was accompanied hy a reduction of the ethanol-induced oxidative su~ss: hydrogen pet*~mde productmn was decreased from 259 • 22 to 149 • 15% tp<0 001) in the 2El cells, and tram t78• 20 to 143 -+ 12% in the Neo cells. In addition, raitochondrial GSH, which was strikingly decreased by ethanol from 1743 • 0.51 to 7 13 ::~ 062 mnol/b~g protein in 2E1 cells (p<O tK)l) and tram 1800 • 0.43 to 1454 • 0.74 in Neo ceils (p<0 01) was parua|ly restored by lycopene to 11 43 • 0.30 (p<O.O1) in 2E1 cdts and to 1 6 2 0 • in Neo cells. The placebo had no protective eitect Conclusion: he potent antioxidant property oI lycopene was corroborated by its striking attenuation of he oxidative stress induced by ethanol in 2El cells, as determined by a reduction in H202

generation and less GSti depletion This was accompanied by a slgniticant attenuation of e hanoi-induced apoptosis The parallehsm between the efiects on oxidauve stress and on apoptosis suggest a causal link If these beneficial effects of lycopene can he reproduced in v~vo, they may provide tbe necessary experimental data to justity a clinical trial of lycopene l~ man

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A Key Pathogenic Role for The Statl-T-Bet Signaling Pathway in Con a-Induced l_iver Injury Juergen Siebler, 5telan Wirtz, Benno Weigmann, Hans A. Lehr, Mune H. Glimcher, Peter R Gall< Markus F Neurath

Backround: Con A injectinn in mice n'sults in an dose dependent liver injury. 1FN-gamma has been suggested to play a role in ConA reduced liver damage The role at cytokine dependent signaling pathways has not been well elucidated, however in the presetu study, we investigated the role of STAT1-T bet signaling pathway by creating STAT1 transgene mice and using STAT1 and f-bet deticient mice Mater~a~ethods The human STAT1 eDNA was cloned downstream of the hCD2 minimal enhancer/promoter into an expression vector >elding the hCD24TAT1 plasmid A 12.7 kb SaiL/Nail fragment containing the 5FATI expre~ion cassette was micromjected into pronudei of fertilized eggs of FVB mice. Mice we~e maintained under specific pathogen-tree conditions m isolated cages Con A was mjected in STAT1 transgenic, STAT1 deficient and T~bet deficient mice (25mg/kg i.e.). 8 hour~ after Con A administration rmce were killed, blood was taken and livers were lixed n t0rma[in. Results: We generated STAT1 transgmnc mice under the control of the T cell specihe hCD2 promoter/enhancer. PCR analysis of the of{);pnng mice demonstrated integra- ton of the construvt in 5 mice Two independent founders (no 2 and 7) were used tar additional experinlents 8h alter ConA injection AUUAST were signihcantly more elevated n STATl-trangenic mice in comparisou to STAT1 wildtype mice, while STAT1 deficient mice revealed normal values T-bet deflcxent mice showed significant lower ALT/AST values ~han l'-bet wild-type control aninx*ls after Con A challenge. Liver sections of STAT 1 transgene mice showed diffuse and degenerative changes while there was no pathological finding in SIArl deficient mice alter ConA injection These degenerative changes were also tound in T-bet wildt-ype mice whereas T-bet deficient mice showed s~ginficantly less pathological changes Discussion: the lz'sults suggest an essential role for the STAT1-T-bet signaling pathway in ~he devdopment of Co*~a_ induced liver damage. Since anti-fEN-gamma antibodms taft to block the nim~ase in nuclear STATI expression after Con A challenge, our data suggest the exlsteme of IFN-gamma independent signaling events that cause STAT1 activation m Con A induced liver injury. Further studies are required to identify such signals that msuh in an activation of the STAT1-T-het signaling pathway in Con A induced liver damage

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HepatoceUular Proliferation Following Chronic Ethanol Exposure Involves TNFe* ~ Induced Activation of H*Ras Matthias Froh, Fuyumi Isayama, Ming Yin, Susanne Netter, Juergen Schoelmerich, MlcheaI D. Wheeler

INTRODUCTION: TNFc~ has been shown to be both pro-apoptotic and -prolifera/ive tar hepatocytes, and is necessary tbr alcohol-induced liver injury. Ras, a knovm pro-oncogene, is very important in the regulation of ce|lnlar responses to TNFe~. AIM: The purpose ol this study was to investigate the state of Ras activation in livers of ethanolded imce METHODS and RESULTS: In male C57B1/6 mice led ethanol (16-20 g/kg/day) via inu-agastric cannulation for 4 weeks, Ras activation was deternmred by pull-down assay using a GST-Rafl Ras binding domain substrate followed by" Western blot tar total Ras or tc~r specific Ras isolorms. Active GTP-bound Ras was increased significantly aher 4 weeks of ethanol. However, in rnice deficient in the receptor ibr TNFc* (TNFRI-/-), the increase in active GTP-bound Ras was siginficandy blunted Using anubodies specific for each isoibrm, it was demonstrated that H-Ras, K-Ras, and N-Ras were increased after ethanol exposure m wild-t)~e nnce. R-Ras was unchanged under these conditions Interestingly, the ethanoI4nduced H-Ras and K-Ras activity was significantly" blunted in in TNFR1-/- ~nice. Ethanol induced N-Ras activation was similar to wild-type Inhibition of H-Ras with adcnovlrus containing a dominant-negauve Ras blunted ethanol-induced hepatocyte proliferatmn by greater than 50%, but had no etlect on ethanol-induced liver injury. CONCLUSION: These data support the h3q~otbesis that Ras activation in response to TNF~ may play" an important role in hepatic regeneration following ethanol-induced liver injury Moreover, TNFa signaling may inwqve specific isoforms of Ras, and these isoforms may have distinct signaling properties, acung as either a protective or toxic mediator during chronic ethanol exposure

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Macrophage Inflammatory Protein-lee (MIP-ler Enhances Experimental Liver Injury Through Induction of Proinflammatory Cytokines in Mice Shiro Yokohama, Yosui Tamaki, Taku ha, Taku Ira, Satoshi Okamoto, Mitsuyoshi Okada, Kazunobu Aso, Masashi Yoneda, Kimihide Nakanmm~ isao Makuta

Recently, it has been reported that chemokines participate the development of immune- mediated liver d i~ses such as vn'al, autoimmune and alcoholic hepatitis Ahhougb CC- chemokines recruit and activate monocytes, macrophages and T cells, the role of CC- chemokines in T-cell mediated-liver inju U still remains investigated Purpose: To nwcstigate a role at MIP- le~, one at CC-ehemokmes m concanavalin A (Con A)-induced 'i"-celI medialed- liver injury in nrice. Methods: In female Balb/c mice (7 weeks old), liver inn U was induced by intravenous injection of Con A (15 or 20 mg/kg/ ~ni-MiP-lc~ antibody (MIP-la Ab; 50 or 200 mghnouse, iv'), recomhinant murme MIP-Ic~ ~recom-MlP-lc< 0.5 or 2 rag/mouse, ip), and gadolinium chloride, a hmctiomal Kupffer cell inhibitor, (GdC1; 40 mg/kg, ip) were administrated pnor to Con A injecuon. Plasma ALT level was determined enzymatically 8 and 24 h after Con A injection. Plasma MIP-lcq TNF-~ and IFN-y levels were determined after Con A rejection by ELISA Liver tissues were obtained 24 h aher Con A ii~}ection and were stained by H&E lbr an assessment of hepatic damage Resuhs: Plasma MIP-lc* level was elevated and roached a peak value at 2 h alter Con A injection The elevated plasma ALT level at 8 hatter Con A ir~}ectinn was dose-dependently inhibited (Mean + - SE, KU/ L: vehicle 7973 +- 882; 50 mg 4326 + ~ 1460; 200 mg 2815 +- 627, n=8) by MIP-Ia Ab pretreatment The necrotic change at midzonal zone of the livers and mortality- rate 24 h after Con A rejection were also reduced by MIP-lc* Ab pretreatment On the other hand, plasnra ALT level at 8 h after Con A rejection were dose-dependently enhanced by' recom- M1P-ln pretreatment. The elevated plasma MIP-lcr level at 2 h and ALI" level at 8 h after Con A injection were significantly inhibited by GdC1 pretreatment The elevated plasma TNF-~ level at 2 h and IFN-y level at 8 h after Con A injection were inhibited by MIP-ly Ab pretreatment. By" contrast, plasma TNF-e~ and IFN-y levels atier Con A injection wet~ significantly increased by recmn-M1P-ic~ pretreatment Conclusmn: These tindings suggest that MIP-I~ is produced fi'om Kupfler ceils after C(m A injection, and this CC chemokitm plays a role in Con A-induced liver injury through induction of proinflammatory cytokmes in mice.

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Role of Nitric Oxide in Hepatic and Pulmonary Injury Jollowing Ischemia- reperfusion of Rat Liver Yuji Takamatsu, Kazoo Shimada, Koji Yamaguchi, Kazuo Chiliiwa, Masao Tanaka

BACKGROUND: The role of nitric oxide (NO) in hepatic isehemiaqeper[dsion (I/R) injury is controversial. NO can be beneficial by improving microcirculation and decreasing leukocyte accumulation, but an excessive production of NO can react with superoxide to produce perox)mitrfte, a highly toxic nrolecule Our objective was to investigate the role of NO produced by reducible NO s~mthase (iNOS) in hepatic I/R injury using a highly selective iNOS inhibitor, ONO-1714 METHODS: SD rats were divided into 2 groups. I/R group was subjected to 90 rain of partial hepatic ischemia tollowed by 3, 6, 12, and 24 h of reperl~.ision. ONO-1714 group was subjected to the same protocol except for the administration of ONO- 1714 (0,03 mg/kg s.c. just befo~ reperfusion and every 6 ha. Expression of iNOS mILNA in the liver and lung was determined by semiqnantilative RT-PCR and plasma nitrate/ nitrite level was examined by Griess metMd. Liver injury was assessed by plasma ALT and histological examination and lung nijury was assessed by histology' and capillary leakage using Evans Blue dye. Localization of peroxynitrite was examined by- the immunohtstochemi- cal detection of nitrotyrosnie. RESULTS: Hepatic I/R induced iNOS mRNA expression with a peak at 3 to 6 h after reperfusion in the liver and lung. Plasma NO level was increased nearly 4 folds at 24 h after reperfusion (42 _+ 5 ~,M, p<0.05 vs sham) ONO-1714 eftectively inhibited this increase. Plasma ALT was peaked at 12 h (2952.0 • 705,9 IU/1) and ONO- 1714 significantly attenuated this increase (701.0 _+ 106.3 IUA), There was no effect of ONO- 1714 on the nlicrovascular permeability o{ the lung. Histological examination revealed extensive necrosis, hemorrhage and sinusoidal congestion in the liver and inflammatory, cell

A-719 AASLD Abstracts