Absence of Linkage between the Retinoblastoma Gene and hts Gene in the LEC Rat: A Model of Human Wilson's Disease

The LEC rat is an authentic model of human Wilson's disease (WD) with an autosomal recessively inherited hepatitis. We investigated linkage between the hepatitis gene ( hts ) and the rat retinoblas-toma gene (RB), that is closely linked to the WD gene in humans, to see whether or not the hts gene is located on the syntenic region of WD and is the counterpart of the WD gene. Polymerase chain reaction-single strand conformation polymorphism analysis with backcross progenies from LEC and TM strains showed that the recombination rate between these two loci was 55.6%, demonstrating that the hts and RB genes are not linked to each other. These data indicate that the hts gene is not the counterpart of the WD gene and that the human syntenic region on which the WD locus and human RB gene are located, is not conserved in the rat genome.     

Abnormal Hepatic Iron Accumulation in LEC Rats

The LEC (Long-Evans cinnamon) rat is a mutant strain displaying hereditary hepatitis and spontaneous hepatocellular carcinoma, and shows abnormal hepatic copper accumulation similar to that occurring in Wilson's disease. We evaluated the iron metabolism of LEC rats compared to LEA (Long-Evans agouti) rats. Hepatic iron and ferritin concentrations were remarkably increased depending on age in LEC rats but not in LEA rats. Increased hepatic iron is normally associated with decreased serum transferrin and total iron binding capacity in hepatic iron overload. In LEC rats, however, both serum transferrin and total iron binding capacity increased with increasing hepatic iron. This increase of serum transferrin and hepatic iron may be an additional important factor contributing to liver injury in LEC rats.     

Evidence of alterations in base excision repair of oxidative DNA damage during spontaneous hepatocarcinogenesis in Long Evans Cinnamon rats

The Long-Evans Cinnamon (LEC) rat, an animal model for Wilson's disease, is an inbred mutant strain, which because of the genetic copper metabolism disorder develops hepatitis approximately 4 months after birth, followed by chronic hepatitis later in life, and eventually all of the surviving animals from liver injury and hepatitis develop spontaneous hepatocellular carcinomas. This animal model also shows that the generation of reactive oxygen species and the accumulation of oxidative damage in the liver DNA has significantly increased over the lifetime of LEC versus the wild-type Long-Evans Agouti (LEA) rats. Thus, the LEC rats having this genetically induced oxidative condition are proved to be very useful model for the study of endogenous DNA lesions and their relation to spontaneous carcinogenesis. In this study, we tested the hypothesis that differences do exist between these two rat strains in respect to their capacity to repair oxidative DNA base modification, which could explain the elevation of endogenous oxidative damage in the LEC rat liver DNA. We found that both the activity and expression at the protein and RNA levels of major DNA glycosylases, endonuclease III and 8-oxoguanine DNA-glycosylase, which initiate the excision and repair of oxidized bases, were significantly altered during the acute (16-18 weeks) and early chronic (24 weeks) phases of hepatitis. Enzyme levels were restored in the later period of chronic hepatitis (week 40) in the LEC rat liver as compared with the age-matched LEA rats. This early reduction in the capacity to repair oxidative DNA base damage could have contributed to the accumulation of mutagenic adducts in liver DNA. These findings show for the first time in an animal model that acute hepatitis impairs the repair of oxidative DNA base damage and strongly suggest that the repair of endogenous DNA adducts plays a critical role in the development of spontaneous hepatocellular carcinoma in LEC rats.     

Genetic linkage between copper accumulation and hepatitis/hepatoma development in LEC rats.

The concentration of copper in the livers of Long-Evans rats with cinnamon-like coat color (LEC), in which hepatitis and then hepatomas develop spontaneously, was recently found to be abnormally high. Therefore, we examined the copper concentrations in the livers of LEC F1 backcrosses (LEC F1 x LEC) to determine the linkage of copper accumulation with development of hepatitis. Consistent with a previously reported ratio of rats with hepatitis to rats without hepatitis of about 1:1, hepatitis developed in 14 of 30 F1 backcrosses. The copper concentrations in the livers of all LEC F1 backcrosses with hepatitis were abnormally high and comparable to those of LEC rats. In contrast, the concentrations in all backcrosses without hepatitis were similar to those in normal Long-Evans with agouti coat color or Brown-Norway rats. Copper accumulation was shown to be closely linked with the development of hepatitis in LEC rats and appeared to be a possible cause of hepatitis. The concentrations of copper in the livers of Fischer 344 rats after carbon tetrachloride treatment were in the range for normal liver, indicating that a high copper concentration in the liver is specific to LEC rats and not a specific characteristic of hepatitis. Furthermore, we found that the size and level of ceruloplasmin mRNA in the livers of LEC rats were the same as those in LEA rats and that the size and level of ceruloplasmin polypeptide in their livers and plasma were almost the same as those in LEA rats. Therefore, these results suggest that the copper accumulation is not due to alteration of expression or to gross alteration of the ceruloplasmin gene.     

Role of Atp7b gene in spontaneous and N-diethylnitrosamine-induced carcinogenesis in a new congenic strain, WKAH.C-Atp7b rats.

To examine whether Long-Evans Cinnamon (LEC) rats, a mutant rat model of Wilson's disease, have a susceptibility gene(s) to hepatocarcinogenesis in addition to the causative gene, Atp7b, we established a new congenic strain, WKAH.C-Atp7b rats, in which the Atp7b gene of the LEC rats is inserted into the normal Wistar-King Aptekman Hokkaido (WKAH) background. Hepatocellular tumors developed spontaneously in both sexes of WKAH.C-Atp7b rats, their incidence being slightly lower than that in LEC rats. Incidences of spontaneous liver tumors in LEC, WKAH.C-Atp7b and WKAH rats correlated with hepatic copper and iron concentrations. Medium-term liver bioassay showed that LEC rats were more susceptible to the induction of glutathione S-transferase placental form-positive preneoplastic foci than WKAH.C-Atp7b rats, and WKAH.C-Atp7b rats were more susceptible than WKAH rats. In an N-diethylnitrosamine (DEN)-induced long-term carcinogenicity study, 1) LEC rats were similarly or rather less susceptible to hepatocellular tumors than WKAH.C-Atp7b and WKAH rats, indicating that the progression of the preneoplastic foci to liver cancer in LEC rats was worse than that in WKAH.C-Atp7b and WKAH rats, 2) the incidences of kidney tumors in LEC and WKAH.C-Atp7b rats were higher than that in WKAH rats and high copper concentrations in the kidneys were observed in LEC and WKAH.C-Atp7b rats, 3) LEC rats were resistant to lung carcinogenesis. These data indicate that the susceptibility of LEC rats to liver and kidney carcinogenesis could be explained by Atp7b gene mutation and that the susceptibility to lung carcinogenesis is controlled by gene(s) other than Atp7b.     

Hereditary low level of plasma ceruloplasmin in LEC rats associated with spontaneous development of hepatitis and liver cancer

Both young (5 weeks old) and old (61-100 weeks old) hereditary hepatitis LEC rats showed a markedly low level of plasma ceruloplasmin (Cp) ferroxidase activity as compared with that of age-matched LEA and BN strain rats. This trait was genetically examined by the use of (BN x LEC) F1 hybrid and (F1 x LEC) backcross rats. The F1 hybrids never developed hepatitis and showed a similar level of Cp to that found in the parental BN rats. Among the backcross rats with about 1:1 segregation rate for hepatitis, affected rats had a remarkably decreased level of Cp, as found in LEC rats, whereas unaffected rats exhibited a similar level of Cp to that of BN, F1 and LEA rats. These results indicate that the low level of Cp is heritable in a single autosomal recessive mode in LEC rats. The observed tight link between the low Cp level and the hepatitis in LEC rats suggests that defective copper metabolism may be associated with the occurrence of hepatitis in LEC rats, since Cp is a copper-binding protein primarily involved in copper transport from the liver.     

Genomic DNA analyses of spontaneous hepatocellular carcinomas in LEC rat liver using a new technique

An inbred rat strain, LEC (long evans cinnamon) has been used as a model of human Wilson's disease. This animal suffers from a severe type of hepatitis, the clinical manifestations of which are similar to human fulminant hepatitis for 4-5 months which is caused by accumulation of copper in the liver. The surviving rats develop chronic hepatitis, followed by the development of spontaneous hepatoma. In contrast to studies with hepatocellular carcinomas (HCCs), the studies have great advantages in that the animals have identical genetic background, can be raised under a fixed condition, and the development of HCC is reproducible. We took two HCC samples and analysed their genomic DNA using RLGS (restriction landmark genomic scanning), which involves two-dimensional electrophoresis of genomic DNA allowing the survey of some 1,000 NotI sites throughout the genome. Using this technique, we discovered landmark spots that were either decreased or increased in intensity in HCC and compared them with the RLGS profile obtained from the DNA of control normal LEC rat liver. Approximately 1,300 spots were compared, and the intensity of two spots was found to be decreased about half and one was increased 1.3-1.7 folds. Although the mechanism of these changes and the properties of the changed DNA are yet to be studied, recurrent genomic changes in the LEC rat HCC could prove to be a good model system for elucidating the essential genetic events in association with hepatocarcinogenesis.     

Role of Atp7b Gene in Spontaneous and N-Diethylnitrosamine-induced Carcinogenesis in a New Congenic Strain, WKAH.C-Atp7b Rats

To examine whether Long-Evans Cinnamon (LEC) rats, a mutant rat model of Wilson's disease, have a susceptibility gene(s) to hepatocarcinogenesis in addition to the causative gene, Atp7b , we established a new congenic strain, WKAH.C-Atp7b rats, in which the Atp7b gene of the LEG rats is inserted into the normal Wistar-King Aptekman Hokkaido (WKAH) background. Hepatocellular tumors developed spontaneously in both sexes of WKAH.C-Atp7b rats, their incidence being slightly lower than that in LEG rats. Incidences of spontaneous liver tumors in LEG, WKAH.C- Atp7b and WKAH rats correlated with hepatic copper and iron concentrations. Medium-term liver bioassay showed that LEG rats were more susceptible to the induction of glutathione S-transferase placental form-positive preneoplastic foci than WKAH.C -Atp7b rats, and WKAH . C -Atp7b rats were more susceptible than WKAH rats. In an N -diethylnitrosamine (DEN)-induced long-term carcinogenicity study, 1) LEC rats were similarly or rather less susceptible to hepatocellular tumors than WKAH.C-Atp7b and WKAH rats, indicating that the progression of the preneoplastic foci to liver cancer in LEG rats was worse than that in WKAH.C-Atp7b and WKAH rats, 2) the incidences of kidney tumors in LEG and WKAH.C-Atp7b rats were higher than that in WKAH rats and high copper concentrations in the kidneys were observed in LEG and WKAH.C- Atp7b rats, 3) LEC rats were resistant to lung carcinogenesis. These data indicate that the susceptibility of LEG rats to liver and kidney carcinogenesis could be explained by Atp7b gene mutation and that the susceptibility to lung carcinogenesis is controlled by gene(s) other than Atp7b.     

Apoptosis and age-dependant induction of nuclear and mitochondrial etheno-DNA adducts in Long-Evans Cinnamon (LEC) rats: enhanced DNA damage by dietary curcumin upon copper accumulation

Long-Evans Cinnamon (LEC) rats, a model for human Wilson's disease, develop chronic hepatitis and liver tumors owing to accumulation of copper and induced oxidative stress. Lipid peroxidation (LPO)-induced etheno-DNA adducts in nuclear- and mitochondrial-DNA along with apoptosis was measured in LEC rat liver. Levels of etheno-DNA adducts (1,N6-ethenodeoxyadenosine and 3,N4-ethenodeoxycytidine) increased with age reaching a peak at 8 and 12 weeks in nuclear and mitochondrial DNA, respectively. This is the first demonstration that etheno-DNA adducts are also formed in mitochondrial DNA. Apoptosis was assessed by TUNEL+ cells in liver sections. CD95L RNA expression was also measured by in situ hybridization in the same sections. The highest nuclear DNA adduct levels coincided with a reduced apoptotic rate at 8 weeks. Mitochondrial-DNA adducts peaked at 12 weeks that coincided with the highest apoptotic rate, suggesting a link of etheno-DNA adducts in mitochondrial DNA to apoptosis. The DNA damage in liver was further enhanced and sustained by 0.5% curcumin in the diet. Treatment for 2 weeks elevated etheno-DNA adducts 9- to 25-fold in nuclear DNA and 3- to 4-fold in mitochondrial-DNA, providing a plausible explanation as to why in our earlier study [Frank et al. (2003) Mutat. Res., 523-524, 127-135], curcumin failed to prevent liver tumors in LEC rats. Our results also confirm the reported in vitro DNA damaging potential of curcumin in the presence of copper ions by reactive oxygen species. LPO-induced adduct formation in nuclear and mitochondrial DNA appear as early lesions in LEC rat liver carcinogenesis and are discussed in relation to apoptotic events in the progression of malignant disease.     

Increased mutant frequency and altered mutation spectrum of the lacI transgene in Wilson disease rats with hepatitis

The mutant strain Long-Evans Cinnamon (LEC) rat, which accumulates copper in the liver because of a mutation in the Atp7b gene, encoding a copper-ATPase, is a model of Wilson disease. It spontaneously develops hepatitis, and subsequently hepatocellular carcinoma and cholangiofibrosis. Excess intracellular copper has been thought to induce DNA damage through reactive oxygen species produced by Cu (II)/Cu (I) redox cycling, and also by direct interaction with DNA. We have developed lacI transgenic Wilson disease (WND-B) rats by mating LEC with Big Blue F344 rats carrying a lambda shuttle vector harboring the lacI gene. lacI mutations of the livers of C-B heterozygous (Atp7b w/m, lacI) and WND-B homozygous (Atp7b m/m, lacI) rats at 6, 24, and 40 weeks of ages were analyzed. Mutant frequencies in the WND-B rats were 2.0 +/- 0.7 x 10(-5), 5.3 +/- 0.9 x 10(-5), and 5.3 +/- 1.0 x 10(-5), respectively, significantly higher than those of C-B rats. Nucleotide sequence analysis revealed that the frequency of deletion mutations of more than two nucleotides were much higher, 15% in WND-B rats, but only 2% in C-B rats. In addition, the average size of deletion was larger in the former. Loss of oligonucleotide-repeat units was specific and relatively frequent in WND-B rats. This type of mutation might be implicated in the induction of DNA strand scissions by reactive oxygen species. These findings suggest that the increase in mutant frequencies and/or the specific type of mutation according to copper accumulation play a crucial role in hepatocarcinogenesis in LEC rats.     

Hereditary Low Level of Plasma Ceruloplasmin in LEC Rats Associated with Spontaneous Development of Hepatitis and Liver Cancer

Both young (5 weeks old) and old (61 100 weeks old) hereditary hepatitis LEC rats showed a markedly low level of plasma ceruloplasmin (Cp) ferroxidase activity as compared with that of age-matched LEA and BN strain rats. This trait was genetically examined hy the use of (BN × LEC) F₁ hybrid and (F₁× LEC) backcross rats. The F₁ hybrids never developed hepatitis and showed a similar level of Cp to that found in the parental BN rats. Among the backcross rats with about 1:1 segregation rate for hepatitis, affected rats had a remarkably decreased level of Cp, as found in LEC rats, whereas unaffected rats exhibited a similar level of Cp to that of BN, F₁ and LEA rats. These results indicate that the low level of Cp is heritable in a single autosomal recessive mode in LEC rats. The observed tight link between the low Cp level and the hepatitis in LEC rats suggests that defective copper metabolism may he associated with the occurrence of hepatitis in LEC rats, since Cp is a copper-binding protein primarily involved in copper transport from the liver.     

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.     

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.     

Gene expression of placental glutathione S-transferase in hereditary hepatitis and spontaneous hepatocarcinogenesis of LEC strain rats

Liver tissues of LEC rats, which develop fulminant hepatitis and hepatocellular carcinoma (hepatoma), were examined by Northern blot analysis using a cDNA probe of rat placental glutathione S-transferase (GST-P). GST-P gene expression was observed not only during hepatocarcinogenesis but also in fulminant hepatitis before development of chronic hepatitis and hepatoma in LEC rats. Cholangiofibrosis in LEC rats also showed high GST-P expression. A transplantable cell line derived from spontaneous LEC hepatoma exhibited a remarkably high expression. By contrast, very weak expression was observed in the livers of young LEC rats before development of hepatitis and control strain rats. Thus, spontaneous hepatic lesions in LEC rats may provide a new clue to elucidate the mechanism of GST-P gene expression.     

Gene Expression of Placental Glutathione S-Transferase in Hereditary Hepatitis and Spontaneous Hepatocarcinogenesis of LEC Strain Rats

Liver tissues of LEG rats, which develop fulminant hepatitis and hepatocellular carcinoma (hepatoma), were examined by Northern blot analysis using a cDNA probe of rat placental glutathione S -transferase (GST-P). GST-P gene expression was observed not only during hepatocarcinogenesis but also in fulminant hepatitis before development of chronic hepatitis and hepatoma in LEC rats. Cholangiofibrosis in LEC rats also showed high GST-P expression. A transplantable cell line derived from spontaneous LEC hepatoma exhibited a remarkably high expression. By contrast, very weak expression was observed in the livers of young LEC rats before development of hepatitis and control strain rats. Thus, spontaneous hepatic lesions in LEC rats may provide a new clue to elucidate the mechanism of GST-P gene expression.     

Pathological comparison between spontaneously developed and chemically induced liver cancers in LEC rats with hereditary hepatitis

To investigate the pathological differences between spontaneously developed and chemically induced liver cancers in LEC rats with hereditary hepatitis, eight-week-old LEC rats were fed 0.06% or 0.03% of 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) for 12 weeks, and then fed basal diet. Spontaneous liver cancers occurred in 60.4% of the LEC rats, while 3'-Me-DAB induced cancers in all LEC rats. The survival periods of chemically (0.06%) treated LEC rats were significantly shorter than non-treated rats. The chemically-induced cancers were strongly related to Edmondson classification. Metastasis and transplantability of the chemically-induced cancers were higher than spontaneous cancers. LEC rats are highly susceptible to the chemical carcinogen with the initiated status of hepatocarcinogenesis.     

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.     

High Sensitivity of LEC Rats with Chronic Hepatitis to Hepatocarcinogenesis: Decreases in Unscheduled and Replicative DNA Synthesis of the Hepatocytes

We carried out the following three experiments to clarify the mechanism of hepatocarcinogenesis in Long-Evans Cinnamon (LEC) rats. (1) Sensitivity to diethylnitrosamine (DEN): LEC rats (8 and 25 weeks old) without and with hepatitis and age-matched F344 rats were administered an intraperitoneal injection of a low dose of DEN. Eight weeks after the injection, the numbers of glutathione-S-transferase placental-form (GST-P)-positive foci in the 33-week-old LEC rat liver were significantly higher than those in the livers of the other three groups of rats. (2) Potential for unscheduled DNA synthesis (UDS): Isolated hepatocytes of 25-week-old LEC rats with chronic hepatitis showed about one-third the level of UDS induced by UV irradiation, as compared to that of age-matched F344 rats, while no significant difference was found between the UDS of isolated hepatocytes of 8-week-old LEC rats and age-matched F344 rats. (3) Potential for proliferation: Isolated hepatocytes from 8-week-old LEC rats responded well to epidermal growth factor (EGF) in culture, to almost the same degree as F344 rat hepatocytes, while a remarkable decrease in the responsiveness of hepatocytes isolated from 25-week-old LEC rats to EGF was found. These results suggested that LEC rat hepatocellular carcinoma could be naturally initiated after the onset of hepatitis by carcinogens contaminating food and the environment, probably due to the reduction of DNA repair activity, after which initiated hepatocytes selectively proliferate in response to growth stimuli endogenously produced as a result of continuous loss of hepatocytes (chronic hepatitis), because of a decrease in growth activity of non-initiated hepatocytes.     

Abnormal Copper Accumulation in Non-cancerous and Cancerous Liver Tissues of LEC Rats Developing Hereditary Hepatitis and Spontaneous Hepatoma

We studied the copper concentrations in the non-cancerous and cancerous liver tissues of LEC rats with hereditary hepatitis and spontaneous hepatoma by atomic absorption spectrophotometry. Copper concentration in the non-cancerous livers of 29-month-old male LEC rats was comparable to that in the livers of LEC rats aged 2, 3 and 8 months whose hepatic copper concentrations were more than 40 times those of normal LEA rats. Copper concentration in spontaneously developed hepatocellular carcinomas of the 29-month-old male LEC rats was lower than that in the surrounding non-cancerous liver tissues, but was still more than 39 times that of 8-month-old male LEA rats. These findings suggest that in LEC rats an abnormal copper metabolism may be maintained during the process of hepatic carcinogenesis.     

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. Morphometric 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.