Hepatic adaptive cytoprotection through the nitric oxide pathway

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• L0329 HETER OGENEITY OF CELL RENEWAL IN MOUSE LIVER: [3H]THYMIDINE AUTO-RADIOGRAPHIC INVESTIGATION. S. Kokuno, Y. Magami, M. Furukawa, Y. Tsukioka, D. Nakayama, T. Azuma, H. Innkuchi, K. Kawai, T. Hattori, T. Saitoh. Dept. of Internal Medicine, Tokyo Medical College Hospital, Tokyo, Dept. of Preventive Medicine, Kyoto Prefectural University of Medicine, Kyoto, Dept. of Pathology, Shiga University of Medical Science, Shiga, Japan. The cell kinetics of liver have not been fully analyzed such as gastrointestinal mucosa. In the rapid renewing cell population, such as the gastrointestinal mucnsa, cell kinetics study can be performed by the short period of labeling with [3H]thymidine. However, in the slow renewing cell population, such as liver, it is difficult to examine the cellular turnover precisely. In the past we reported on the cell kinetics of mouse hepatocytes in this meeting. Furthermore, we studied zonal difference of cell renewal of hepatocytes in liver. We also compared cell renewal of hepatocytes with that of ductal cells. In order to study the cell renewal, we used the pulse labeling method after the long continuous labeling with [-~H]thymidine. The animals received 112 repeated injections of [3H]thymidine at 6hr. intervals for 28 days after birth and were killed on 1st, 90th, 200th, and 300th days after the last injection. Immediately after killing the animals, the liver was fixed and embedded in paraffin. The sections were dipped in nuclear emulsion and developed after 5 weeks exposure and finally stained with hematoxylin and eosin. After 112 repeated continuous labeling, more than 90% of the parenchymal cells in the liver were labeled. This finding indicates that most of cells in liver arise in postnatal days. Mean grain counts of the labeled hepatocyte were 35.6, 31.9, 25.4, and 18.4 on lst., 90th, 200th, and 300th day, respectively. The mean grain counts decreased to half for 297.1 days. The mean grain counts of ductal cells decreased more rapidly than those of hepatocytes. This indicates that ductal cells show a high rate of cell renewal in comparison with hepatocytes. We studied the spatial distribution of labeled hepatocytes in liver. Mean grain counts of hepatocytes in perihepatic vein region decreased more rapidly in comparison to those in periportal vein region. This finding indicates that perihepatic vein region may be proliferative zone in liver. These study indicate that liver is not homogeneous but heterogeneous on its cell renewal rate. L0330 TOCOPHEROL ACETATE PROTECTS AGAINST HEPATIC INJURY AFTER ISCHEMIA-REPERFUSION: EVIDENCE FOR A ROLE FOR SUPEROXIDE FREE RADICALS. H. K0matsu, Y. Sajima, K. Imamura, H. Masuda, K. Nishiyama, Y. Shinomiya, T. Sakai, T. Tsuchihashi, H. Ishii. Yokohama Municipal Citizen's Hospital, Yokohama, Japan, and Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan. Ischemia-reperfusion injury of the liver is a serious problem in liver transplantation. We have shown in ischemic, reperfused rat liver that an accumulation of neutrophils occurs and superoxide free radicals play a role in mediating this neutrophil accumulation (Am. J. Physiol., 262: G669-G676, 1992). Tocopherol acetate (vitamin E) functions as the major membranelocalized cellular antioxidant, protecting membrane phospholipids from peroxidative damage initiated by free radicals. Serum and hepatic vitamin E is particularly prone to deficiency in the patient with chronic cholestatic liver disease. The aims of this study were to study whether or not vitamin E would decrease liver neutrophil content and hepatic injury after ischemiareperfusion. Methods: In fasted, Nembutal-anesthetized (50mg/kg) male Wistar rats (300-350g), ischemia-reperfusion was induced by clamping the left branches of hepatic artery and portal vein for 25 min, then declamping and sacrificing the animal 24 h later. Vitamin E (10mg/kg) was given by intramuscular administration 1, 3 and 5 days prior to the experiment. Myeloperoxidase (MPO) activity of liver as a marker for nentrophil content, serum GPT and histologic necrosis score were measured as previously reported (Hepatology, 15: 507-514, 1992). Results: MPO Serum GPT ( 1 U / L ) Histologic (U/g liver) 0 min 24h Necrosis Score Control 131.8 ± 9.5 49 ± 7 485 ± 73 1.3 ± 0.3 Vitamin E 91.6 ± 7.7* 45 ± 6 341 ± 30* 0.7 ± 0.2* (Mean ± SEM, *p < 0.05, ANOVA followed by Dunnett's test) Conclusion: Pretreatment with vitamin E significantly decreased the elevated liver MPO activity, serum GPT and the extent of hepatocellular necrosis, thus confirming a role for superoxide anion in the pathogenesis of hepatic reperfusion injury and the clinical therapeutic value of vitamin E. This study was supported in part by a grant from the Ministry of Welfare.

GASTROENTEROLOGYVol. 114, No. 4 • L0331 TELOMERASE ACTIVITY OF NEEDLE-BIOPSIED LIVER SAMPLES: ITS USEFULNESS FOR DIAGNOSIS AND EFFICACY JUDGEMENT OF TREATMENT OF SMALL HEPATOCELLULAR CARCINOMA. F K0mine, M Moriyama, M Shimojima, Y Arakawa; 3rd Dept. Of Internal Medicine, Nihon University School of Medicine, Tokyo, Japan. The discovery of small hepatooellular carcinoma (HCC) related to chronic hepatitis C has recently increased in Japan and its pathological diagnosis is not always easy. The aim of this study is evaluate a clinical usefulness of the measurement of telomerase activity for the needle biopsied samples which were obtained for the diagnosis of small HCC and the judgement of treatment efficacy. Materials and Methods The subjects investigated were 15 patients (19 nodules), who were found to have intrahepatic tumorous nodules, less than 20mm in size, by abdominal ultrasound(US) between April and November, 1997. Fifteen patients were chronic hepatitis C with or without cirrhosis. All the patients underwent needle biopsies for both the tumorous nodules and extranodular regions of livers. The samples underwent histopathological diagnosis and the measurement of telomerase activity by the fluorescence-based telomeric repeat amplification protocol method. Furthermore, re-biopsy of the same nodule was performed one week after the treatment of percutaneous ethanol injection therapy (PELT) to measure telomerase activity for 7 patients (7 nodules), who were diagnosed to have HCC. As a control, the liver biopsy of 30 patients with chronic hepatitis were measured for telomerase activity. Results Eight nodules were pathologically diagnosed to have extremely well-differentiated HCC and exhibited a high telomerase activity (mean 35.1U). Among them one HCC with the largest size and having a focus of less differentiated HCC exhibited the highest activity (85.2U). On the other hand, I1 nodules were diagnosed as atypical hyperplasia (borderline lesion). Among them 5 nodules showed a low telomerase activity and the remaining 6 showed the activity of 0.0U (mean 8.7U). All the nontumorous hepatic portions of the 15 patients exhibited a mean of 2.8U and the control livers exhibited 0.0U. The telomerase activity increased statistically significantly in a stepwise fashion from non-tumorous lesion, atypical hyperplasia and finally to well-differentiated HCC. The PElT resulted in the loss (0.0U) of telomerase activity in 6 patients and its persistence in 1 patient. Discussion. The present investigation clearly shows that the measurement of telomerase activity is useful for the final diagnosis of intrahepatic tumorous nodules and the assessment of efficacy of PElT treatment. The persistence of high activity after the PEIT treatment may indicate the failure of complete elimination of HCC, necessitating strict follow-up. Also, the difference of telomerase activity seems to justify the histopathological diagnosis of atypical hyperplasia and HCC. L0332 ENDOSCOPIC MUCOSAL ELECTROCOAGULATION FOR THE TREATMENT OF RECURRENT ESOPHAGEAL VARICES. Y. Konishi, T. Nakamura, K. Okazaki, T. Chiba. Division of Gastroenterology and Hepatology, Department of Medicine, Postgraduate School of Medicine, Kyoto University, Kyoto Japan. Background and Study Aims: Some patients with esophageal varices resist endoscopic treatments such as endoscopic variceal ligation(EVL) or endoscopi c injection sclerotherapy(EIS). In these cases, varices are small and hard to ligate, and repeated EIS might cause severe complications. To overcome these problems, we developed endoscopic mucosal electrocoagnlation therapy(Emec). Patient and Methods: Ten patients with recurrent small esophageal varices, all who had a history of endoscopic treatment for variceal hemorrhage, were treated by Emec. We achieved electrocoagulation within the lower 5 cm of the esophageal mucosa using a bipolar probe. Results: During an average of 13.8 months after Emec, nine patients (90.0%) had no sign of recurrence. We found pyrexia only on the treatment day in 8 of 10 patients as the most frequent complication. No other severe complication was recognized. Conclusion: We developed Emec and obtained excellent results in the treatment of recurrent esophageal varices. Emec can be an alternative therapy for recurrent small varices. • L0333 HEPATIC ADAPTIVE CYTOPROTECTION THROUGH THE NITRIC OXIDE PATHWAY. T. Kono, M. Yoneda, N. Ando, K. Kamiya, K. Ohara, H. Karasaki, Y. Yamada, Y. Yamamoto, H. Kotani, K. Kikuchi-Utsumi, J. Iwamoto and S. Kasai. Dept. of Surgery II, Medicine II and Physiology II, Asahikawa Medical College, Asahikawa, Japan. Background: It has been proposed that nitric oxide (NO) plays a major role in the pathogenesis of lipopolysaccharide (LPS)-induced liver damage, however there is no consensus on the function of NO in the liver. A very low dose of LPS (below 0.01 mg/k~,, i.p.) does not induce liver damage but elicits a large production of NO in the liver (FASEB J., 97). This phenomenon is contradictory to the aspect of cytotoxicity of NO. Aims: To explore the significance of hepatic NO production induced by a low dose of LPS in regard to a possible cytoprotective effect. Methods: Wistar rats (n=60) were injected a low dose of LPS (0.001 mg/kg, i.p.), and then at 1, 4, 8, 10, 16, or 24 hours, they were injected a large dose of LPS (5 mg/kg, i.p.), which is known to cause severe hepatotoxicity. Ten hours later, the animals were sacrificed and

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April 1998 the magnitude of liver damage was assessed by measuring serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and by histopathological examination. Inducible nitric oxide synthase (iNOS) mRNA expression in the liver was demonstrated by Northern analysis and NO released from the liver into the abdominal cavity was measured by chemiluminescence. Inhibitors of iNOS, aminoguanidine (25 mg/kg, i.p.) or S-methylisothiourea (SMT; 12.5 mg/kg, i.p.), was co-injected with the first LPS injection. Results: Ten hours after the single high dose ol~ LPS (5 mg/kg), a marked increase of AST and ALT (Mean -+ SE: AST; 805 +- 117, ALT; 720 ___52) was observed. The low dose of LPS (0.001 mg/kg) injected 8 hours before the high dose of LPS, attenuated the elevation of serum AST and ALT levels (AST; 131 ~ 5, ALT; 87 + 10). This cytoprotective effect was first observed when the high dose of LPS was injected 4 hours after the low dose LPS and the maximal effect was 8 hours. With longer intervals, this cytoprotective effect was gradually weakened. The cytoprotection induced by the low dose of LPS was reversed by the co-injection of aminoguanidine or SMT. On the other hand, the low dose of LPS induced iNOS mRNA expression in the liver. This was noticed at 60 min and it peaked at 4 hours after the injection, then it gradually decreased thereafter. No NO was detected in the abdominal cavity before LPS injection, but it became detectable at 4 hrs after LPS injection and it reached its maximum level at 8 hours (900 -+ 148 ppb) without any signs of associated liver damage. Conclusions: These results suggest a very low dose of LPS can induce "hepatic adaptive cytoprotection" against high dose LPS-induced liver damage via the NO pathway. This work was supported by a grant-in-aid (A)(2) 07407030 from the Ministry of E.S.C.of Japan.

significantly increased in the first 48 hours following CCl4exposure in the IL-6 -/- livers. To determine whether pretreatment with IL-6 prior to CC14exposure had any effect on liver injury, hepatocyte apoptosis, and recovery, IL-6 -/- and IL-6 +/+ mice were injected with a single subcutaneous dose of recombinant IL-6 (rlL-6) followed in 20 minutes by a single intraperitoneal dose of CC14. The IL-6 -/- mice pretreated with rlL-6 (IL-6 -/- + rlL-6) had reduced liver injury noted histologically at 12, 24, 36, and 48 hours post- CClacompared to IL-6 4- livers. The IL-6 +/+ mice pretreated with rlL-6 (IL-6+/+ + rlL-6) had reduced injury compared to all groups. There was a significant decrease in hepatocyte apoptotic cell death in the IL-6 -/- + rlL-6 livers at 12, 24, 36 and 48 hours post- CClacompared to the IL-6 -/- livers. There was a significant decrease in hepatocyte apoptotic cell death in the IL-6 +/+ + rlL-6 livers at 24 and 36 hours post- COl 4 compared to the IL-6 +/+ livers. These findings were confirmed by the TUNEL assay. Treatment with rlL-6 also accelerated hepatocyte recovery as measured by DNA synthesis and mitosis. Bromodeoxyuridine incorporation was significantly increased in both groups treated with rlL-6 at 36 hours postCCl 4. Mitotic events were significantly increased at 36 hours post- CCI4 in the IL-6 +/+ + rlL-6 livers. IL-6 +/+ livers demonstrated increased STAT3 and NF-~:B binding. STAT3 and NF-~:B binding was greatly reduced in the IL-6 4- livers. Treating the IL-6 -/- mice with rlL-6 normalized STAT3 binding and corrected NF-~zB binding. Our data suggests that IL-6 is a protective factor against toxic liver injury. IL-6 may derive at least some of its ability to protect the liver through its demonstrated antiapoptotic effects. NF-KB is a potent anti-apoptotic factor and inducer of IL-6. Our data suggests that at least some of the anti-apoptotic effects of NF-~:B may result from its ability to stimulate IL-6 gene expression.

L0334

HIGH RATE HAEMOSTASIS IN ACTIVE BLEEDING FROM ESOPHAGEAL VARICES TREATED WITH SCLEROTHERAPY + SOMATOSTATIN IV. A.Konstantinidis, A.Germanopoulos, E.Kanellopoulou, V.DelIs, V.Balatsos, A. Pantes, V.Vamvakousls, V.Ekonomou, J.Karuballs, J.Drikos, N.Skandalls Gastroenterologist Unit,General Hospital "G.Gennimatas", Athens, Greece. Aim: To show the efficacy of sclerotherapy plus somatostatin iv, in active bleeding from esophageal varrices. Patients: 202 (146 male, 56 female,aged 17-93 years) with 264 episodes of active bleeding from esophageal varrices. 156 p. suffered one episode of active bleeding, 34 p. two, 8 p. 3, and 4 p. four episodes. The causes of cirrhosis-portal hypertension were : alcohol 72, HBV 43 (+ alcohol 6, + 81, +C 3), HCV 38 (+ alcohol 5), PBC 13, PV Thrombosisi 7, cryptogenic 29. According to Child-Puhg scale they were classified as : A 71, B 85, C 46 patients. Method: Urgent UGI endoscopy, assesment of bleeding varrices and scierotherapy with EO 5% and continous infusion of somatostatin 3 mg / 12h iv for 2-5 days. Active bleeding = spurting or oozing bleeding from varrix, or a blood clot adhered to a varix or blood in the stomach without another prominent site of bleeding. Failure of haemostasis = continuing haemorrhage (shock, haematemesis/melena for > 2-3 days, decrease of the Hct after stabilization, need for blood transfusions > 4-6 units) or relapse within 5-10 days. Results: Haemostasis was successful in 172 out of 202 p.(85.1%) or in 209 of 262 episodes of varriceal bleeding (79.8%). In 30 p. the haemorrhage continued for > 2-3 days or relapsed within 5-10 d after the 1 rst session of treatment. 21 of them were further treated with more Scl + Sum. sessions whereas in 9 of them the bleeding was refractory to any treatment (3 died and 6 of them were subsequently oeperated, of whom 3 p. successfully. The overall rate of succeful treatment was 95.5 % (in 193 p or in 234 of 262 episodes of bleeding 89.3%). The average blood units transfused were 4.3 per patient. Complications such as retrostemal pain, fever, pneumonia, pleuritic effusion were conservatvety treated. CONCLUSION: Sclerotherapy + Sum. iv, in active esophageal varriceal bleeding, is ultimately successful in 85-95 %. In 15-25 % of the episodes in which haemorrgage continues or relapses in 10 days, the repeated 2nd-3rd session succeeds in 70 % of cases. • L0335 TREATMENT WITH INTERLEUKIN-6 REDUCES LIVER DAMAGE AND APOPTOTIC HEPATOCYTE DEATH AND ENHANCES RECOVERY IN ACUTE TOXIC INJURY. K. Knvaiovich. E.E. Furth, R. Taub. Departments of Genetics, Pathology, and Medicine; Division of Gastroenterology, University of Pennsylvania School of Medicine, Philadelphia, PA. Determining what factors are responsible for ameliorating injury and promoting recovery in response to acute liver injury are central issues in understanding the molecular basis of hepatic regeneration. Our laboratory recently demonstrated that liver regeneration after partial hepatectomy is dramatically impaired in mice with a targeted disruption of the intefleukin-6 gene (IL-6 -/-) verses controls (IL-6 +/+). Regeneration is corrected in IL-6 -/- mice with a single pre-operative dose of IL-6. We also noted that IL-6 -/- mice develop more rapid and more severe hepatocellular injury following acute carbon tetrachloride (CC14) exposure. There is a significant blunting of both the DNA synthetic response and mitotic response to CC14in the IL-6 -/- livers reflecting impaired recovery. Hepatocyte apoptosis is

L0336

PORTAL BLOOD FLOW MEASURED WITH DUPLEX DOPPLER ULTRASOUND CORRELATES HISTOLOGICAL ACTIVITY OF CHRONIC LIVER DISEASE IN CHILDREN. Kosuke Kozaiwa. Atsushi Sawada, Hitoshi Tajiri, Yoko Miyoshi, Kanae Tada, Yuri Etani, Kazunori Miki. Shintaro Okada. Department of Pediatrics, Osaka University, Faculty of Medicine, Osaka Japan.

[Back~ruundl We reported on an utility of measurement of the maximal velocity of the blood flow in the main portal trunk (Vmax) in evaluating chronic liver disease in children (J Pediatr Gastroenterol Nutr 1995; 21: 215-9). However, there are several practical difficulties in measuring Vmax; experienced skill is needed to gain the maximal velocity: bowel gas and obesity may cause poor imaging of the main portal trunk. [Aim] To investigate a correlation between Vmax and histological activity index (HAl) score in chronic liver disease and to explore another parameter in addition to Vmax which is easier to assess and useful in evaluating chronic liver disease. [Methodsl Portal blood flow was studied in 102 children with chronic liver diseases. Velocity was measured at the main portal trunk (Vmax), the umbilical portion of the left portal branch (UP), the right portal branch (RP), and the splenic vein in the splenic hilus (SV). Eighteen of them were evaluated both prior to and after treatment. The ratio of thickness of the lesser omentum/diameter of the abdominal aorta at the level of the origin of the celiac axis (Ld/Ao) was also studied with B-mode ultrasound in 41 patients. Examinations were performed in the afternoon following a 4-h fast with supine position using Sonolayer SSH-160 system (Toshiba) with a Doppler frequency of 3.75 MHz. Resistive index (R.I.) of hepatic artery was also evaluated in 15 patients. Liver histology was examined in 60 cases. [Resultsl Significant negative correlation was observed between Vmax and total HAI score (p<0.01). Periportal necrosis score (p<0.05) and fibrosis score (p<0.05) showed significant correlation with Vmax. No correlation was observed between intralobular degeneration or portal inflammation and Vmax. After treatment Vmax increased significantly in responding patients (p<0.05). Significant negative correlation was also observed between Vmax and Ld/Ao (p<0.001). Six patients with normal Ld/Ao ratios showed a decreased low Vmax, five of them showing active liver disease. Velocity of RP also correlated well with Vmax (P<0.01). Velocities of UP or SV, or R.I. of hepatic artery showed no correlation with Vmax. Significant negative correlation was found between R.I. and HAl total score. Score of portal inflammation correlated with R.I. [Conclusions] Vmax value was shown to be decreased as the pathological stage of liver disease progressed and significantly correlated with periportal necrosis and fibrosis. Both velocity of RP and Ld/Ao are shown to also be useful in evaluating chronic liver disease although Vmax appeared to be the most sensitive among sonographic indicies of liver histological activity.