A l 1 3 0 AASLD ABSTRACTS GASTROENTEROLOGY, Vol. 108, No. 4

• The clinical significance of apoptosis in small hepatcellular carcinoma 'examined by apoptosis related markers' R. Nakashio. M. Kitamoto, S. Suemori*, K. Tsuji, S. Kira, Y. Watanabe, T. Nakanishi, and G. Kajiyama. First Dept. of Internal Medicine, Hiroshima University School of Medieine,Hiroshima, Japan *Gastrointestinal Unit, Yokohama City Seibu Hospital, St. Marianna University School of Medicine, Kanagawa, Japan

The mechanism of carcinogenesis of the liver is unknown, but it is well known that in chronic liver disease or liver cirrhosis hepatcellular carcinoma (HCC) is frequently detected in the process of hyperplastic nodules. In the model of HCC, it was indicated that most of hyperplastic nodules oceiarred in the liver tissue, disappeared by apoptosis and the residue changed to carcinoma. Many apoptotie cells occurred in the liver tissue were information on the duration of the histologically visible stage. In this study, we examined apoptosis in the non tumor tissue sections from small HCC patients and compared the conditions of apoptosis with their recurrence after resection. Methods: Formalin fixed and paraffin embedded tissue sections were obtained from 22 patients of HCC less than 3cm in diameter. We stained the sections immunohistochemically by using LevisY antibody (BM1, it was claimed to be closely correlated with apoptosis), and stained DNA fragmentation patterns (ApopTag, a marker of apoptosis) by using in situ hybridization method, called nick end labeling (NEL) technique. We microscopically examined non-tumor liver tissue around the main tumor of these sections. LevisY antibody stained cytoplasm positively, and NEL stained nuclear positive,: Results: 7 of 22 sections were stained by both of LevisY antibody and NEL technique in the same area of non-tumor liver tissue. They were thought to be occurring apoptosis. 5 of 7 sections were stained in interlobular and periportal area, 2 of 7 sections were stained in periportal area alone. On the other hand, 15 of 22 sections were not stained by LevisY antibody or NEL technique in the same area of non-tumor liver tissues. They were not thought to be occurring apoptosis. All of 7 patients with apoptotsis-positive sections recurrented within 3 years after resection. 7 of 15 of patients apoptosis-negative sections did not recurrent. Conclusions: Frequent appearance of apoptosis in non-tumor liver tissues might be a high risk factor of recurrence after resection.

PROLONGED DECREASE IN HEPATIC CONNEXIN32 IN CHRONIC LIVER INJURY INDUCED BY CARBON TETRACHLORIDE IN RATS. Y.Nakata, MSato, Y.Watanabe, S.Kimura. Depts. of Surg.ll, Ehime Univ.School of Medicine.

Gap junctions are intercellular channels which allow the passage of small molecules. Cell-to-cell communication through gap junctions is involved in growth control, embryonic differentiation, and the propagation of neural signals between hepatocytes. The major component of gap junctions in rat liver is connexin32 (CXN32). The content of gap junctions in the liver is known to be decreased in relation to hepatocyte proliferation. However, CXN32 content in the liver is transiently reduced in acute liver damage after single administration of carbon tetrachloride (CC14) without an increase in DNA synthesis. This observation suggests that there exists a different mechanism regulating connexin content from that observed during hepatocyte prolifera!iou. It thus appears interestingly to examine whether repeated and prolonged liver injury causes the changes in connexin content in rats.

The alteration of CXN32, was studied in rats after chronic administration of CCI4. Animals were injected with CCI4 (0.5mllkg) twice a week for 12 weeks. The activity of alanine-aminotrausferase in plasma increased from 30 U/I to about 1000 U/1 after 12 weeks, but recovered nearly to normal level in 7 days after cessation of CC14 injection. Liver specimens 12 days after the last injection of CC14 appeared cirrhotic with a marked increase in fibrosis. Conuexin32 contents in these livers, determined by immunoblotting, decreased to about 37% of controls. Significant decrease in connexin32 content was retained at least for 30 days and recovered to the control level by 60 days. Such an alteration of connexin32 content in chronically injured liver was confirmed by immunohistochemical examination. These results suggest that intercellular communication through gaP junctions is disturbed in chronic liver injury, lasting even after recovery from the acute phase of injury. In addition, since the level of connexin32-mRNA was not reduced, but rather increased by chronic injection of CCI4, the prolonged decrease in connexin32 content in chronically injured liver might be due to a derangement of the post-transcriptional control.

• VASOPRESSIN REDUCESTAUROCHENODEOXYCHOLATE- INDUCED HEPATOTOXITY BY LOWERING TAUROCHENODEOXY- CHOLATE CONTENT IN HEPATOCYTE T. NAKAZAWA, M. HOSHINO, T. HAYAKAWA, Y KAMtYA, T. YAMADA, H. OHHara, K.MIZUNO, H. YAMADA, T. INAGAKI, O.MIYAJI, T.TAKEUTI: First Dept. of Internal Medicine. NagoyaCity University, Schoolof Medicine. Nagoya, Japan.

We have reported that taurochenodeoxychoUc acid(TCDCA) content in hepatocyte was proportional to its hepatotoxicity using release of LDH activity from primary cultured rat hepatocytes in vitro. Futhermore, we have found that tauroursodeoxycholic acid(TUDCA) and dbcAMP reduced both TCDCA-induced hepatotoxiclty and intracellular TCDCA levels by accelerating the efflux of intracellular TCDCA( Hepatology1993;17:470-476. Scand J Gastroentero11993;28:833-838.). In contrast, papaverine increased both TCDCA-induced hepatotoxicity and intracelluar TCDCA content by a reduction of vesicular transport system in hepatoeyte(Hepatology 1994;20:692-699.). Vasopressin(VP) has been reported to reduce bile flow by many investigators, but the effect of VP on bile acid secretion and hepatotoxicity is still unclear In the present study, we investigated the influence of VP on TCDCA-induced hepatotoxicity and biliary excretion of TCDCA in primary cultured ra!. hepatocyte and isolated pertused rat liver(IPRL). ' 1) The simultaneous addition of VP with lmM TCDCA significantly decreased the release of LDH in proportion to the increase of VP concentration as compared with lmM TCDCA given alone and also reduced the intracellular levels of TCDCA from 19.3 + 1.0 to 13.0 _+ 0.7 nmol/mg protein. 2) Affer 30 min culture with lmMTCDCA, and rinsing with a medium free of bile acid, recuituring hepatocytes in a medium without TCDCA gradually decreased the intracellular levels of TCDCA. The presence of VP (10 9 M) in the reculturing medium emphasized this disappearance speed. 3) Superimposition of VP (100 pmot/L) on TCDCA infusion at a constant rate of 1.0 ,umol/min(25 ,umol/L)~resulted in quick increases in bile flow and biliary excretion of bile acids (6~.5 + 4.9 vs 3.5 _+ 16.7 nmol/g, liver) as calculated by numerically integrating the area under the curve, and significantly reduced the increase in release of LDH from the perfused liver 4) One-minute loading of HRP(25 mg) produced an early(4 to 6 min) and late(20 to 25 rain) peak of biliarY .excretion of HRP. The continuous infusion of VP(330 pmol/L) significantly increased both early peak (defined as O to 10 min) and late peak (defined as 10 to 50 rain), from 1.83 _+ 0.31 and 8.48 + 0.46 to 4.11 + 0.89 and 21.7 + 2.70 ng/gliver, respectively . We concluded that VP has a cytoprotective effect against TCDCA-induced hepatotoxicity by stimulating the secretion of TCDC in part via vesicular transport system in hepatocytes.

• ALTERED BILE ACID PROFILE 1N DIET-INDUCED PIGMENT STONES. A. Nakeeb, K. Fox-Talbot, P.A. Lipsett, K.D. Lillemoe, H.A. Pitt. The Department of Surgery, The Johns Hopkins Medical Institutions, Bakimure, Maryland

High carbohydrate, low protein diets have been shown to cause pigment gallstones in hamsters and prairie dogs. In these models, biliary levels of bilimbin and calcium are increased leading to calcium bilirubinate precipitation. Bile salts may also play a role in bilirubin sohbilization; however, little is known about the effect of these pigment stone diets on individual bile acid compositiOn. Therefore, we tested the hypothesis that a high carbohydrate, low protein pigment stone diet would aker the individual bile acid mih'eu. Twent-one eight week old male Syrian Golden hamsters (SASCO) were fed either a control non-lithogenic (CTL;n=I 1) or a high carbohydrate, low protein, nutritionally complete pigment stone diet (PIG,n = 10) for 20 weeks. Gallbladders were examined for the presence of pigment stones, and hepatic bile was collected on ice in light protected tubes for two hours. Bile was analyzed for biliary lipids by standard techniques and for individual bile acids by reverse phase HPLC. Pigment stones were identified in 70% of PIG and 36% of CTL hamsters. Results for total lipid content (TLC), total bile acids (TBA), and for cholic acid iCA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), ursodeoxycholic acid (UDCA) and lithocliolic acid (LCA) were:

TLC(~cn/dI) TBA(pM/ml) CA(%) CDCA(%) DCA(%) UDCA(%) LCA(%)

CTL 1.5±0.1 22.2±3.4 65.6±1.7 20.7±1.6 9.7±1.2 3.1±0.6 1.0=:0.2

PIG 1.0±0.1. 12.4±1.4~ 54.8±1.6" 26.0±1.2J- 9.2±0.9 7.6±0.5* 2.3±0.5~ Mean ±SEM * p <0.01i "1" p<0.05 vs controls (Student's T- test)

These data suggest that the high carbohydrate, low protein pigment stone diet 1) lowers total lipid content, total bile acid, and cholic a~id levels and 2) increases chenodeoxycholic, ursodeoxycholic and lithocholic acid levels in hepatic bile. The reduced bile acid concentrations and altered profile may alter the ability of bile salts to keep bilh'ubin in solution and thereby contribute to pigment stone formation in this model. We conclude that the level and distribution of individual bile acids may play a role in diet induced pigment gallstone path0geaesis.