Kimimaro Dempo

High Susceptibility to Hepatocellular Carcinoma Development in LEC Rats with Hereditary Hepatitis

A remarkably high incidence of hepatocellular carcinomas was observed in long‐surviving LEC rats with hereditary hepatitis. Among the 60 LEC rats examined between 12 and 28 months of age from F29, and F30, 55 (92%) developed putative preneoplastic and neoplastic lesions such as hyperplastic foci and nodules, and hepatocellular carcinomas. Of these, hepatocellular carcinomas were observed with a high frequency (46/55; 84%). All rats of advanced age that survived more than 18 months developed hepatocellular carcinomas. These results suggest that the development of liver tumors in LEC rats is an age‐associated phenomenon with serial hepatic alterations after the subsidence of acute hepatitis. The long‐surviving rats had no normal tissue and showed chronic hepatitis in nontumorous tissues of the liver. Cholangiofibrosis was also found in most rats with hepatic lesions. Metastasis of hepatocellular carcinomas was found in four rats. Histologically, the hepatocellular carcinomas were of a well‐differentiated type with a typical trabecular structure. Thus, LEC rats seem to be a promising animal model for studying the pathogenesis of hepatitis and hepatocellular carcinoma.

SPONTANEOUS HEPATITIS IN LONG-EVANS RATS: A Potential Animal Model for Fulminant Hepatitis in Man

Spontaneous hepatitis associated with severe jaundice occurred in 90% of an inbred strain of Long‐Evans rats. The rapidly progressive syndrome was characterized by abrupt onset, hyperbilirubinemia and increased serum levels of glutamic oxaloacetic transaminase and glutamic pyruvic transaminase, associated with massive and multifocal necrosis of the liver. This strain should provide a useful animal model for analysis of the pathogenesis of fulminant hepatitis in humans. ACTA PATHOL JPN 38: 1369–1375, 1988.

α-FETOPROTEIN AND EARLY HISTOLOGICAL CHANGES OF HEPATIC TISSUE IN DAB-HEPATOCARCINOGENESIS*

It has been generally accepted that the sequential histological changes observed in the hepatic tissue of rats during the course of hepatocarcinogenesis are as follows: Diffuse hyperplasia of hepatocytes appearing in the initial stage, nodular proliferation of the cells in the following stages, and hepatomas in the final stage.’. ’’ During these carcinogenic courses, stepwise alteration of hepatocytes leading to cancer might occur as an expression of preneoplastic transformation. One of the phenotypic changes in preneoplastic stages of hepatocarcinogenesis is the appearance of muscle-type isozyme of aldolase, as in hepatomas.” This type of enzyme was further identified as a fetal one? It is conceivable that isozymes and/or isoproteins in preneoplastic cells as well as in neoplastic cells are of the fetal type. The pattern of serum protein synthesized by hepatocytes is expected to shift from adult to fetal type as the cells undergo cancerization. It was reported that a-fetoprotein (AFP) , one of the fetal serum proteins, was found in a certain period of hepatocarcin~genesis.~-~) The authors have studied hepatic changes of rats administered with 3’-Me-DAB, and obtained the following results: l”-l‘, 2!) ( 1 ) The period of AFP appearance was coincident with that in which a deviation in isozyme pattern of various enzymes of the tissues toward fetal and neonatal types was observed. These enzymes were aldolase, acid phosphatase, and nonspecific esterases. ( 2 ) In the liver, there were a great number of so-called “oval cells,” some of which showed images of differentiation toward hepatocytes. ( 3) Oval-cell proliferation and its differentiation to the hepatocytes may play a principal role in the occurrence of fetal deviation in the function of the hepatic tissue during the early stages of carcinogenesis.

Accumulation of Abnormally High Ploid Nuclei in the Liver of LEC Rats Developing Spontaneous Hepatitis

Enlarged hepatocytes with huge nuclei were found in LEC rats with hereditary hepatitis. Flow cytometric analysis of the DNA content of nuclei from jaundiced LEC rats revealed the presence of very high polyploids, such as 32n and 64n. At the age of 12 weeks, before the onset of hepatitis, 8n polyploid nuclei were more frequent in LEC rats than in LEA rats, a sibling line of LEC rats. Binucleated hepatocytes were also more frequent in LEC rats than in LEA rats at week 4. Bi‐, tri‐ and tetra‐nucleated cells whose nuclei were sometimes different in size were observed when jaundice became manifest. The number of proliferating liver cells, determined by pulse labeling with 5‐bromo‐ 2′‐deoxyuridine (BrdU), was higher in LEC rats than in LEA rats at 2, 4, 8, 12 and 14 weeks, with a maximum at week 4. A remarkable increase of BrdU uptake was observed at week 16, when jaundice developed. The possible involvement of abnormal cytokinesis and kariokinesis in the manifestation of hepatitis was suggested.

Elevation of serum alpha-fetoprotein and proliferation of oval cells in the livers of LEC rats.

Alpha‐fetoprotein (AFP) in the sera of 35 LEC (Long‐Evans with a cinnamon‐like coat color) rats between 7 and 25 weeks of age was evaluated by enzyme‐linked immunosorbent assay (ELISA). Elevation of serum AFP and proliferation of oval cells in the liver were observed in most LEC rats, which suffered from acute hepatitis. On the other hand, the serum AFP level was within the normal range before the onset of hepatitis. Immunohistochemical staining for AFP revealed that some of the proliferating oval cells produced AFP, Morphotnetric analysis of AFP‐positive cells and ELISA for serum AFP demonstrated that there was a statistically significant correlation between the number of AFP‐positive cells in the liver and the concentration of AFP in the serum. Histological examination revealed the transition and differentiation of the oval cells to small hepatocytes. These results suggested that the phenomena which occurred in LEC rats suffering from acute hepatitis were similar to those that occurred during the early stage of azo dye hepatocarcinogenesis, although the extent of the oval cell proliferation and the elevation of serum AFP in LEC rats were not as great as those in rats treated with azo dye. This is the first report on a rat strain with proliferation of AFP‐producing oval cells during its natural history.

Progress from Chronic Hepatitis to Liver Cancer in Long-Surviving LEC Rats

In our previous papers, we reported on the clinical and pathological characteristics [1], pathogenesis [2], and combined immunodeficiency of spontaneous hepatitis in LEC rats.

High Susceptibility to Spontaneous Development of Hepatocellular Carcinoma in LEC Rats

Spontaneous hepatitis was first found in the LEC strain rat, which had been maintained conventionally at the Center for Experimental Plants and Animals, Hokkaido University [1]. The hepatitis appears suddenly in LEC rats at about 4 months after birth, and leads to a high mortality [2]. Characteristics of the disease are similar to those of fulminant hepatitis in humans, showing bilirubinuria, hyperbilirubinemia (frequently severe jaundice), subcutaneous bleeding, loss of body weight, increased levels of serum glutamic oxaloacetic transaminase, glutamic pyruvic transaminase and alkaline Phosphatase, and single-cell necrosis or spotty necrosis of liver tissues with faint inflammatory cell response. Genetic analysis by crossing tests indicated that the disease has an autosomal recessive mode of inheritance, being ruled by a single gene designated hts (symbol for hepatitis) [3]. Many rats died of submassive necrosis in liver tissues within 1 week after the onset of hepatitis. The remaining animals survived with chronic hepatitis more than 1 year and developed preneoplastic and neoplastic lesions of the liver [2]. Electron microscopic analysis failed to reveal any viral particles in the affected liver, and intraperitoneal injections of liver homogenates of jaundiced rats did not induce hepatitis in neonatal rats of another strain [2], although viral hepatitis has been reported to be associated with hepatocarcinogenesis in humans [4, 5], woodchucks [6], ground squirrels [7] and ducks [8]. Although the cause of hepatitis in LEC rats is different from that of viral hepatitis in the other cases, regenerative stimuli caused by hepatitis may promote development of liver tumors in all cases. In this report, the natural history of spontaneous hepatic lesions in LEC rats are described with regard to the anatomical and histopathological aspects. Furthermore, establishment and characterization of a hepatocellular carcinoma (hepatoma) cell line obtained from an LEC rat are reported.

Clinical and Pathological Characteristics of LEC Rats with Spontaneous Hepatitis

Currently, there are very few available animal models for the research of human hepatitis. We have known for many years that the marmoset and the chimpanzee are sensitive to hepatitis A and B viruses, respectively. It has been reported that the woodchunk (Marmota monax) shows a high incidence of persistent infection with woodchuck hepatitis virus (WHV), one of the hepadna viruses, and that the incidence of hepatocellular carcinomas in WHV-positive woodchucks is extremely high [1].

Induction of a Novel Ca2+-Dependent Serine Protease in Rat Liver Treated with Various Promoters of Liver Carcinogenesis

The induction of a novel Ca2+-dependent protease in rat liver treated with various liver promoters, as well as its increase in preneoplastic lesions during liver carcinogenesis, was demonstrated. Six groups of male Fischer 344 rats (150 g body weight) were fed separately diets containing one of the following promoters: 0.05% phenobarbital (PB), 0.05% dichlorodiphenyltrichloroethane (DDT), 0.25% ethyl-α-chlorophenoxyisobutyrate (CPIB), 0.5% butylated hydroxytoluene (BHT), 10 ppm 17-α-ethynylestradiol (EE), and 0.05% of the non-promoter diphenylhydantoin (DH). After feeding the indicated diets for 1 week, rats were killed and protease activity in the microsomal fraction of liver tissue was determined using N-benzoyl-L-tyrosine ethyl ester as substrate. The activity of protease increased 3- to 5-fold after treatment with the promoters and compared with normal liver; the non-promoter (DH) induced a slight increase in activity. Hyperplastic nodules were induced according to the method of Solt and Farber. The activity of protease was significantly high in these preneoplastic lesions compared with the surrounding liver tissue. Biochemical characterization of this protease revealed the following properties: 1) high Ca2+ dependency, 2) different molecular weight and optimum pH from previously reported proteases, and 3) preferential distribution in the SER fraction. These results suggest that a novel type of protease is induced specifically in the liver by promoters of liver carcinogenesis. The possible importance of this protease in the carcinogenic process is discussed.

Combined Immunodeficiency in LEC Rats with Spontaneous Hepatitis

One of the abnormalities in the LEC rat is immunoglobulin G (IgG) formation, and a definite correlation has been found between suppressed IgG formation and the incidence of hepatitis [1, 2]. Therefore, we investigated both cellular and humoral immunological competence in relation to the incidence of hepatitis.