HEPATOLOGY Vol. 34, No. 4, Pt. 2, 2001 AASLD ABSTRACTS 519A

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MUTATIONAL CHANGES IN CYP 2C19 GENE IN DRUG-INDUCED LIVER DISEASES. Ryukichi Kumashiro, Akiko Hisamochi, Yuriko Koga, Tat- suya Ide, Kei Ogata, Shiro Murashima, Teruko Hino, Kurume University, Ku- rume city Japan; Takato Ueno, Kurume University Center for Innovative Can- cer Research, Kurume city Japan; Michio Sara

d~im: Cytochrome P450 (CYP) is involved in phase I drug metabolizing sys- tem. In Japanese population, the homozygous mutat ion (poor metabolizer) for this enzyme is reported to be about 18%. This study aims at wheather muta- tional changes of this enzyme is related to liver diseases caused by various drugs; Methods: We analized mutational changes in CYP2C19 gene in patients with drug-induced liver diseases. Patients were devided into 7 groups accord- ing to the suspected causative drugs.Group A is 15 pattens in whom drugs that affect metabolic pathway such as troglitazone, acarbose, pioglitazone, pravas- ratine, bezafibrate, and fenofibrate were used. Group B is 11 patients in whom drugs that affect central nervous system such as chlorpromazine, sulpiride, valproic acid, diazepam, and tiaprid were used. Group C consisted of 8 aptients in whom suspeted causative agents were antibiotics, tamoxifen, autonomic nerve agents, and histamine H2 blocker. Group D were 8 patients in whom the causative drugs were hard to identify. Genotyping of CYP 2C19 was performed w~Lth a polymerase chain reaction (PCR) using peripheral leukocytes. Results: Polymorphysm observed for CYP 2C19 were * 1 (wild), *2 and *3. In group A, CfP2C19 wild ( ' 1 / ' 1 ) , heterozygotes (either *1/ '2 or " 1 / ' 3 ) , and homozy- gores (either *2/*2 or *2/*3 or *3/*3) were 20 %, 47% and 33%, respectively. In group B, wild, heterozygotes and homozygotes were 28 %, 36% and 36%, respectively. In groups C and D, we found no homozygous mutation. The inzidence of homozygous mutat ion for CYP2C19 were higer in patients in group A and B than in groups C and D. Conclusion: The results of this study shows that mutational changes in CYP 2C19 gene is related to the evolusion of drug-induced liver injury, especially agems that affect metabolic pathway and central nervous system. Whether this is resulted from the nature of drug predisposition, or duration of use needs to be clarified.

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THE EFFECT OF URSODEOXYCHOLIC ACID ON CULTURED HEPATO- CYTES. Michiko Yoshii, Mariko Endo, Sachiko Nakagawa, Ken Shiraishi, Ya- suko Ichinose, Mikiko Miyazaki, Kayoko Ueda, Kumatoshi Ishihara, Koichiro Ozawa, Dept of Pharmacotherapy, Grad Sch of Med Sciences, Hiroshima Univ, Hiroshima Japan

Ursodeoxycholic acid (UDCA) has been used as safe and effective medicine in chronic liver diseases, especially primary biliary cirrhosis by improving both clinical manifestations and bio- chemical parameters. However, on the other hand, there are studies that histological improvement was not established in spite of improvement of biochemical parameters. And the reasons haven't been cleared. In this study, after 2 kinds of cultured hepatoeytes were treated with UDCA, metab- olites of UDCA, ursodeoxychoIyl-taurine (TUDC), or -glycine (GUDC), cytotoxicity of UDCA, TUDC and GUDC, the morphological effects of them, and the effects of them on apoptosis were evaluated. Methods: Clone 9 cells (rat normal liver) or Hep-G2 cells (human hepatoma) were cultured in the 96 well cell culture cluster for study (1) or the 24 well cell culture cluster for study (3) or on the cover glass for study (2) over night. And then they were used for following study (1)-(3). (1) Cytotoxtcity: After cultured cells were treated with UDCA, TUDC, or GUDC for 30 minutes, the activity of lactate dehydrogenase (LDH) released into the buffer were determined w/th LDH test Wako. (2) Morphology: After cultured cells were treated with UDC& TUDC, or GUDC for 30 minutes, ceils were labeled with a fluorescent phospholipid analogue, 2-(6-(7- nitrobenz-2-oxa-13-diazol-4-yl) amino) hexanoyl~l-hexadecanoyI-sn-glycero-3-phosphocholine (NBD C6-HPC) followed observed with a confocal laser scanning microscopy. (3) Apoptosis: Cultured hepatocytes treated with UDCA, TUDC, or GUDC were detected mitochondrial mem- brane potential with DePsipher Fluorometric Mitochondrial Permeability Assay Kit. Results: (1) Relative toxicity on clone 9 cells started increasing at the concentration of 0.1ram UDCA, and 1 mM GUDC, but 1raM UDCA, and 5raM GUDC showed highest relative toxicity, and then relative toxicity was decreased again at more than lmM UDCA, and 5raM GUDC. Relative toxicity. TUDC treated clone 9 cells didn't show LDH lerease (Fig. 1). UDCA~ GUDC, and TUDC treated Hep-G2 cells didn't show LDH lerease. (2) The morphological change of clone 9 cells were observed at more than 0.5raM UDCA, and more than 1 mM GUDC. (3) Mitochondrial membrane potentials of clone 9 or Hep-G2 cells weren't disappeared after treatment of less than 1raM TUDC, G13~9C, and 0.5mMUDCA. [Discussion] The treatment with high concentration of GUDC, UDCA, didn't make clone 9 or Hep-G2 cells release LDH. But the morphological changes of both cells were observed. These results suggest that dramatic decrease of biochemical parameters with UDCA treatment in chronic liver diseases patients may not be equal to morphological improvements. And it suggests that hepatocytes are in high concentration of UDCA and/or GUDC should be averted.

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EXPRESSION OF THE HEPATIC RENIN ANGIOTENSIN SYSTEM IN CHRONIC HEPATITS C. Julie R Jonsson, Department of Surgery, University of Queensland, Brisbane, QLD Australia; Andrew D Clouston, Department of PalLhology, University of Queensland, Brisbane Australia; Peter A Gochee, Eliz- abeth E Powell, Department of Surgery, University of Queensland, Brisbane, QJ_.D Australia

Transforming growth factor-~31 (TGF-131) plays a dominant role in the devel- opment of fibrosis. In cardiac and renal fibrosis, TGF-/31 production is en- hanced by angiotensin II (AII), the principal effector molecule of the renin- angiotensin system (RAS). We have previously shown that the angiotensin converting enzyme (ACE) inhibitor, captopril, significantly attenuates the de- velopment of hepatic fibrosis in the rat bile duct ligation model (Gastroenter- ology in press, 2001). However, the role of the RAS in human hepatic fibrosis has not been delineated. In order to address this, we studied the mRNA ex- pression of renin, angiotensinogen (Aogen), ACE, and the angiotensin type 1 receptor (ATIR) as well as TGF-/31 in liver tissue from patients with chronic hepatitis C (HCV, n=52) and in non-diseased liver (NDL, n = 13) using semi- quantitative realtime RT-PCR. Gene expression was evaluated relative to the housekeeping genes ]3-actin and glyceraldehyde-3-phosphate dehydrogenase and reported as arbitrary units. Renin, Aogen, ACE and AT1R were readily detectable in human liver tissue. There was a 9-fold increase in the expression of ACE in liver tissue from HCV compared with NDL (8.2 2 1.5 vs 0.920.2, p<0.0001). TGF-~31 mRNA expression was also increased in HCV compared with NDL (1.920.3 vs 0.6-+0.2, p=0.0005) with a trend for higher expression of TGF-/31 in livers with fibrosis compared with those with no fibrosis. More- over, TGF-131 expression was highly correlated with ACE expression (r = 0.78, p<0.0001). The results indicate that the components of the RAS are all ex- pressed locally in human liver tissue. The increased expression of ACE and its correlation with TGF-/31 expression supports a role for the RAS in hepatic fibrosis associated with chronic HCV.

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INVESTIGATION OF TISSUE TRANSGLUTAMINASE IN TRANSDIFFER- ENTIATING RAT HEPATIC STELLATE CELLS. Claudia SchnabeI f, Insti- tute of Clinical Chemsitry and Pathobiochemistry, Aachen Germany; Katja Breitkopf f, Birgit Lahme f, Axel M Gressner m, Institute of Clinical Chemistry and Pathobiochemistry, Aachen Germany

Background: Tissue transglutaminase (tTG) catalyzes the formation of ~/-glu- tamyl-E-lysine crosslinks within or between proteins entailing a stabilization of extracellular matrix. The role of this enzyme during liver fibrogenesis is not yet well investigated. Besides the function mentioned above it is suggested to link the latent TGF-Ifl binding protein (LTBP) covalently to the ECM. LTBP is part of the large latent TGF-[3 complex, consisting of a TGF-/3 dimer and its propeptide latency associated peptide (LAP). This covalent binding to the ECM is thought to be an imperativ intermediate step in the activation of TGF-~8. Materials and methods: Hepatic stellate cells (HSC) were isolated from normal rat liver by pronase-collagenase perfusion technique and were cultured on plastic dishes entailing a spontaneous gradual transdifferentiation to myofibroblasts (MFB). On day 7 of culture ceils were trypsinized and reseeded to obtain fully differ- entiated MFBs. MFBs were investigated on day 3 or 4. Transdifferentiation was verifed microscopically and by determination of the upregulation of smooth muscle a-actin in immunomorphologic investigations and in western blot analysis, tTG was investigated by northern and western blot analysis and semi- quantitative Cell-ELISA. Results: In the Cell-ELISA an upregulation from day one to day 2 of about 3.5 fold and to day 7 or 8 of about 5 to 10 fold was found. Accordingly the western blot analysis showed an increased expression during transdifferentiation. Also in northern blot analysis an increased quantity of mRNA could be shown. On protein level no further upregulation of tTG in MFB compared to 7 or 8 days old HSC was found. Conclusion: The results demonstrate an upregulation of tTG during transdifferentiation on mRNA and protein levels. Thus, the enzyme may play a role in hepatic fibrogenesis in vivo and may offer a possible approach to handle fibrogenesis.