HEPATOLOGY Vol. 34, No. 4, Pt. 2, 2001
MUTATIONAL CHANGES IN CYP 2C19 GENE IN DRUG-INDUCED LIVER DISEASES. Ryukichi Kumashiro, Akiko Hisamochi, Yuriko Koga, Tatsuya Ide, Kei Ogata, Shiro Murashima, Teruko Hino, Kurume University, Kurume city Japan; Takato Ueno, Kurume University Center for Innovative Cancer Research, Kurume city Japan; Michio Sara
THE EFFECT OF URSODEOXYCHOLIC ACID ON CULTURED HEPATOCYTES. Michiko Yoshii, Mariko Endo, Sachiko Nakagawa, Ken Shiraishi, Yasuko Ichinose, Mikiko Miyazaki, Kayoko Ueda, Kumatoshi Ishihara, Koichiro Ozawa, Dept of Pharmacotherapy, Grad Sch of Med Sciences, Hiroshima Univ, Hiroshima Japan
d~im: Cytochrome P450 (CYP) is involved in phase I drug metabolizing system. In Japanese population, the homozygous mutation (poor metabolizer) for this enzyme is reported to be about 18%. This study aims at wheather mutational 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 according to the suspected causative drugs.Group A is 15 pattens in whom drugs that affect metabolic pathway such as troglitazone, acarbose, pioglitazone, pravasratine, 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~Ltha 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 homozygores (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 mutation 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.
Ursodeoxycholicacid (UDCA) has been used as safe and effectivemedicine in chronic liver diseases, especiallyprimary biliary cirrhosis by improvingboth clinicalmanifestationsand biochemicalparameters.However,on the other hand, there are studies that histologicalimprovement was not establishedin spite of improvementof biochemicalparameters.And the reasons haven't been cleared.In this study, after 2 kinds of cultured hepatoeyteswere treated with UDCA,metabolites of UDCA, ursodeoxychoIyl-taurine(TUDC), or -glycine (GUDC), cytotoxicityof UDCA, TUDC and GUDC, the morphologicaleffectsof them, and the effectsof them on apoptosiswere 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 wellcell culture cluster for study (3) or on the cover glass for study (2) over night. And then they were used for followingstudy (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-(7nitrobenz-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 hepatocytestreated with UDCA, TUDC, or GUDC were detected mitochondrialmembrane potential with DePsipherFluorometricMitochondrialPermeabilityAssayKit. Results: (1) Relativetoxicityon clone 9 cells started increasingat the concentrationof 0.1ram UDCA,and 1 mM GUDC,but 1raMUDCA,and 5raM GUDCshowedhighest relativetoxicity, and then relative toxicitywas decreasedagainat more than lmM UDCA,and 5raM GUDC. Relativetoxicity.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 morphologicalchange of clone 9 cells were observedat more than 0.5raMUDCA,and more than 1 mM GUDC. (3) Mitochondrialmembranepotentialsof clone 9 or Hep-G2 cellsweren't disappearedafter treatmentof less than 1raMTUDC, G13~9C,and 0.5mMUDCA. [Discussion]The treatmentwith high concentrationof GUDC,UDCA,didn't make clone 9 or Hep-G2 cells releaseLDH. But the morphologicalchangesof both cellswere observed. These results suggestthat dramatic decreaseof biochemicalparameterswith UDCAtreatment in chronic liver diseasespatients may not be equal to morphologicalimprovements.And it suggests that hepatocytesare in high concentrationof UDCAand/or GUDCshould be averted.
... I ,.8
I I I -4 -3 ~ -t I ~ , ~ n c m m tBn:E R,ml. '11~ ,k'.'tL-'~'d I ¢ I;¢1"1~ ,:rl I.I3..I !it 11~1~- 'l~tl ct o'1~ !t c,~tl i~ I ~ : l ~ t u~: ~ C I T ~ 100 = L I ~ leJ,e;~: ~11~'!'¢n36100 ~
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, Elizabeth E Powell, Department of Surgery, University of Queensland, Brisbane, QJ_.D Australia
INVESTIGATION OF TISSUE TRANSGLUTAMINASE IN TRANSDIFFERENTIATING RAT HEPATIC STELLATE CELLS. Claudia SchnabeI f, Institute 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
Transforming growth factor-~31 (TGF-131) plays a dominant role in the development of fibrosis. In cardiac and renal fibrosis, TGF-/31 production is enhanced by angiotensin II (AII), the principal effector molecule of the reninangiotensin system (RAS). We have previously shown that the angiotensin converting enzyme (ACE) inhibitor, captopril, significantly attenuates the development of hepatic fibrosis in the rat bile duct ligation model (Gastroenterology 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 expression 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 = 5 2 ) and in non-diseased liver (NDL, n = 13) using semiquantitative 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. Moreover, 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 expressed 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.
Background: Tissue transglutaminase (tTG) catalyzes the formation of ~/-glutamyl-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-Iflbinding protein (LTBP) covalently to the ECM. LTBP is part of the large latent TGF-[3complex, 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 differentiated 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 semiquantitative 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.