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.     

Increased expression of c-jun gene during spontaneous hepatocarcinogenesis in LEC rats

We have studied the expressions of nine proto-oncogenes (c-myc, N-myc, c-fos, C-jun, p53, H-ras, N-ras, c-raf, hst) and two other genes (PCNA, GST-P) during the spontaneous development of hepatocellular carcinomas (HCCs) in LEC rats. Expression of c-myc, H-ras, N-ras, C-raf, p53 and PCNA genes was detected, but this did not significantly change during the development of HCCs in LEC rats. Expression of N-myc and hst genes was not detectable. Expression of c-fos gene was detected in one HCC case out of four. Significantly increased expression of c-jun gene was observed in the liver tissues of LEC rats aged 8 months. This high expression was decreased with the development of HCCs. On the other hand, the expression of GST-P gene increased in parallel with the clinical course of the development of HCCs in LEC rats. The pattern of c-jun mRNA augmentation was different from that of GST-P mRNA. These observations suggest that c-jun gene may play a role in the spontaneous development of HCCs in LEC rats.     

Spontaneous occurrence of placental glutathione S-transferase-positive foci in the livers of LEC rats

Spontaneous occurrence of placental glutathione S-transferase (GST-P)-positive foci was observed in the livers of 5-month-old LEC rats. Quantitative studies revealed that GST-P foci appeared after the onset of hepatitis. The number and size of GST-P foci increased with age and more foci were induced in males than in females. No sex difference, however, was found in the incidence of hepatitis. Although hepatitis is necessary for the induction of GST-P foci, it is insufficient for their further growth. Since hereditary hepatitis first appears at around 4 months of age, leading to a high incidence of hepatocellular carcinomas in later life, the spontaneous occurrence of the foci may be related to the development of hepatocellular carcinoma.     

SPONTANEOUS OCCURRENCE OF PLACENTAL GLUTATHIONE S-TRANSFERASE-POSITIVE FOCI IN THE LIVERS OF LEC RATS

Spontaneous occurrence of placental glutathione S-transferase (GST-P)-positive foci was observed in the livers of 5-month-old LEC rats. Quantitative studies revealed that GST-P foci appeared after the onset of hepatitis. The number and size of GST-P foci increased with age and more foci were induced in males than in females. No sex difference, however, was found in the incidence of hepatitis. Although hepatitis is necessary for the induction of GST-P foci, it is insufficient for their further growth. Since hereditary hepatitis first appears at around 4 months of age, leading to a high incidence of hepatocellular carcinomas in later life, the spontaneous occurrence of the foci may be related to the development of hepatocellular carcinoma.     

DNA damage triggers imbalance of proliferation and apoptosis during development of preneoplastic foci in the liver of Long-Evans Cinnamon rats

The mutant strain Long-Evans Cinnamon (LEC) rat accumulates copper, resulting in spontaneous hepatitis and subsequent development of hepatocellular carcinomas (HCCs) in the liver, providing a promising model for investigation of the relationship between hepatitis induced by oxidative stress and hepatocarcinogenesis. We examined DNA strand breaks in peripheral blood cells and p53 expression in livers during acute and chronic hepatitis in LEC rats, along with preneoplastic lesions, and cell proliferation and apoptosis in non-cancerous portions of livers from LEC rats aged 7-115 weeks. Immunohistochemistry using antibodies against glutathione S-transferase placental-form (GST-P), proliferating cell nuclear antigen (PCNA), and in situ DNA nick labeling (TUNEL) were used. Long-Evans Agouti (LEA) rats, a sibling line of the LEC strain, were used as controls. In the LEC rats, DNA strand breaks and expression of p53 were significantly higher than that of LEA rats at 24 weeks of age. The number of GST-P-positive (GST-P+) foci/cm2 increased and peaked at 48 weeks old, and the areas rapidly expanded thereafter. The level of cell proliferation increased with the development of hepatitis and was highest at about 48 weeks old. The induction of apoptosis in LEC rats was transiently higher than that in LEA rats during the period from 24 to 34 weeks of age. However, the ratio of PCNA-positive cells to the apoptotic index showed a growth imbalance in favor of cell proliferation, supporting sustained net growth in LEC rats. These findings suggest that DNA damage, reflected in DNA strand breaks, plays a critical role in the development of hepatocellular preneoplastic foci, with an imbalance between high proliferation and relatively low apoptosis.     

用镭射激光捕获技术研究LEC鼠p53基因突变和肝癌发生的关系

目的：研究p53基因突变与原发性肝癌发生发展的关系。方法：利用LEC鼠（10ng evan cinnamon）作为肝癌的动物模型。选取15、37、101周的LEC鼠17、15和12只,正常对照SD大鼠15只,取出肝脏组织进行切片。切片一部分用于谷胱甘肽S-转移酶（GST—P）免疫组化染色,另一部分利用镭射激光捕获技术（laser capture microdissection,LCM）定位切割,进行p53基因外显子5、6／7、8突变的分析。结果：37周龄LEC鼠和101周龄LEC鼠GST-P阳性率高于对照组（P〈0．05）,15周龄LEC鼠GST-P阳性率与对照组相比差异无统计学意义,P〉0．05。p53基因在GST—P表达阳性肝炎组有3例突变（3／12）,GST—P表达阳性肝癌组有12例发生突变（12／17）,肝癌组显著高于肝炎组,P〈0．05。结论：p53突变发生在肝癌形成的早期阶段,随着发展过程,突变率明显增高。GST—P虽然特异性不强,但是不失为一种灵敏的判断肝损伤的指标。     

Enhancing effect of a choline-deficient diet on alterations of hepatic drug-metabolizing enzymes in hepatitis- and hepatoma-predisposed rats (LEC rats)

Marked alterations of hepatic drug-metabolizing enzymes were observed in hepatitis- and hepatoma-predisposed rats (LEC rats) fed a choline-deficient diet. The diet enhanced the development of hepatitis with severe jaundice. The levels of two major classes of cytochrome P-450, P-450PB and P-450MC, were markedly decreased. GST-Yp was dramatically increased, whereas GST-Ya, Yb1 and Yb2 were decreased. LEA rats (the control rats to LEC) fed a choline-deficient diet mimicked LEC rats fed a normal diet in terms of the above enzyme alterations, indicating that hypomethylation is involved in the pathogenesis of hepatitis and hepatoma in LEC rats. Such hypomethylation may initiate the hepatocytes that spontaneously develop hepatitis and hepatoma.     

High expression of heparin-binding EGF-like growth factor in rat hepatocarcinogenesis

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a member of the EGF family and is highly expressed in hepatoma tissues but not in normal liver. However, it is unknown when HB-EGF is induced during hepatocarcinogenesis and what are the mechanisms underlying its high expression in hepatoma. To address this issue, the expression of HB-EGF was investigated during hepatocarcinogenesis in LEC (Long-Evans with a cinnamon-like coat color) rats, which spontaneously develop hepatitis and hepatoma. LEA (Long-Evans with an agouti coat color) rats were used as controls. Furthermore, the induction of HB-EGF mRNA by various agents was investigated in a rat hepatoma cell line and hepatocytes in primary culture. Expression of HB-EGF mRNA in the liver was very low at the stage of acute and chronic hepatitis and markedly increased at the stage of hepatoma in LEC rats. Non-involved tissues adjacent to hepatoma showed low expression of HB-EGF mRNA. Immunochemical studies revealed positive staining in hepatoma tissues. Induction of HB-EGF mRNA by several growth factors was observed in a hepatoma cell line but not in normal hepatocytes. Our results suggest that HB-EGF is associated with the early progression steps of hepatoma. © 1996 Wiley-Liss, Inc.     

Enhancing Effect of a Choline–deficient Diet on Alterations of Hepatic Drug–metabolizing Enzymes in Hepatitis– and Hepatoma–predisposed Rats (LEC Rats)

Marked alterations of hepatic drug–metabolizing enzymes were observed in hepatitis– and hepatoma–predisposed rats (LEC rats) fed a choline–deficient diet. The diet enhanced the development of hepatitis with severe jaundice. The levels of two major classes of cytochrome P–450, P–450_PB and P–450_MC, were markedly decreased. GST–Yp was dramatically increased, whereas GST–Ya, Ybl and Yb2 were decreased. LEA rats (the control rats to LEC) fed a choline–deficient diet mimicked LEC rats fed a normal diet in terms of the above enzyme alterations, indicating that hypomethylation is involved in the pathogenesis of hepatitis and hepatoma in LEC rats. Such hypomethylation may initiate the hepatocytes that spontaneously develop hepatitis and hepatoma.     

High sensitivity of the LEC rat liver to the carcinogenic effect of diethylnitrosamine

The initiation sensitivity of the liver of the LEC (Long-Evans with a cinnamon-like coat color) rat, a new mutant strain with a high incidence of spontaneous liver tumors, was studied by treatment with low doses of diethylnitrosamine (DEN) coupled with modified Solt and Farber's selection. LEC and control LEA (Long-Evans with an agouti coat color) rats received i.p. injections of 10 mg/kg of DEN, then selected by feeding with a diet containing 0.02% 2-acetylaminofluorene (AAF) for 7 days combined with partial hepatectomy (PH). The numbers of placental glutathione S-transferase (GST-P)-positive foci in the livers of LEC rats were 10 times higher than those in LEA rats. These results suggested a high sensitivity of the LEC rat liver to the carcinogenic effect of DEN. The association between initiation sensitivity and spontaneous liver-tumor development and the possible usefulness of the LEC rat for in vivo short-term tests of hepatocarcinogens are discussed.     

Possible involvement of c-myc but not ras genes in hepatocellular carcinomas developing after spontaneous hepatitis in LEC rats.

LEC (Long-Evans with a cinnamon-like coat color) rats develop hepatocellular carcinomas (HCCs) spontaneously. We examined mutations of codons 12, 13, and 61 of the Ha-ras, Ki-ras, and N-ras genes in four HCCs by the polymerase chain reaction (PCR)-single-stranded DNA direct sequencing method. No ras gene mutations were observed, suggesting that ras activation is not involved in spontaneous hepatocarcinogenesis in LEC rats. The expression of mRNAs for c-myc, Ha-ras, c-raf, and the protein phosphatase 2A alpha gene (PP-2A alpha) was also examined in the four HCCs by northern blot analysis. Three of the four HCCs had c-myc expression levels approximately 30-fold higher than that in the liver of control Long-Evans rats with an agouti coat color (LEA), a sibling line of LEC rats, while the remaining HCC had an expression level sevenfold higher than that of control. In contrast, the expression levels of the Ha-ras, c-raf, and PP-2A alpha genes were the same as those in the livers of control rats. Studies of c-myc expression and mitotic index in five other HCCs, two hyperplastic nodules, and two nontumorous portions of livers of HCC-bearing LEC rats that had chronic-phase hepatitis suggested that the high level of c-myc gene expression was not due only to increased cell proliferation but might possibly be more integrally involved in hepatocarcinogenesis.     

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.     

Expression of DNA methyltransferase in LEC rats during hepatocarcinogenesis

The Long-Evans with a cinnamon-like color (LEC) rat is a mutantof the Long-Evans strain that develops hereditary hepatitisand hepatoma with ageing. Age-related changes in the mRNA expressionof DNA methyltransferase (DNA MTase) were examined in liversof LEC rats using Long Evans with an agouti color (LEA) ratsas controls. A dramatic increase in the expression of this mRNAwas observed in LEC rats at 20 weeks when acute hepatitis appeared.Their high mRNA levels were maintained until 52 weeks of age.The mRNA expression as well as DNA MTase activities were foundto be higher in cancer lesions than in adjacent normal tissue.These increases may be related to liver regeneration and toearly events in cellular transformation of 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.     

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.     

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.     

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.     

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.     

Two-dimensional Electrophoretic Analysis of Hepatitis-associated Polypeptides in Liver of LEC Rats Developing Spontaneous Hepatitis

High-resolution two-dimensional polyacrylamide gel electrophoresis in combination with silver staining was used to analyze between 800 and 1000 cytosolic and particulate polypeptides from age-matched livers of normal male Long-Evans rat with Agouti coat color (LEA) and Long-Evans rat with Cinnamon-like coat color (LEC) rats with hereditary trait of hepatitis at ages long before, immediately prior to, and just after the onset of hepatitis. Although the electrophoretic patterns of polypeptide expression were very similar with respect to the overall spot patterns, a number of polypeptides which differed either qualitatively or quantitatively were noted. Two constitutively expressed cytosolic polypeptides, P29.5 (M_r 29.5 kDa/pI 6.73) and P30 (30 kDa/6.70), were not detected in livers of LEC animals at any age. In the normal LEA rats both P29.5 and P30 were detected as early as one day after birth and both were expressed at similar concentrations at all ages. In the LEC rats P30-C (30 kDa/6.68) was constitutively expressed in close proximity to the expected position of P30, and P30-C was not detected in the LEA rats. By means of non-equilibrium pH gradient electrophoresis two relatively basic polypeptides were detected in the LEC rats. P18ne was detected immediately prior to and P27ne immediately after the clinical manifestation of hepatitis. Experiments in F₁ backcross ([LEA × LEC] × LEC) animals, however, failed to demonstrate any genetic link between either the expression or lack of expression of P29.5, P30, P30-C, or P18ne and hepatitis development. P27ne was detected in all backcross animals exhibiting hepatitis, but was never observed in LEC rats prior to the onset of hepatitis. Although we were unable to identify any unique loss of expression of polypeptides which are genetically linked to hepatitis susceptibility in LEC rats, specific subsets of quantitatively modulated polypeptides were detected.     

Two-dimensional electrophoretic analysis of hepatitis-associated polypeptides in liver of LEC rats developing spontaneous hepatitis

High-resolution two-dimensional polyacrylamide gel electrophoresis in combination with silver staining was used to analyze between 800 and 1000 cytosolic and particulate polypeptides from age-matched livers of normal male Long-Evans rat with Agouti coat color (LEA) and Long-Evans rat with Cinnamon-like coat color (LEC) rats with hereditary trait of hepatitis at ages long before, immediately prior to, and just after the onset of hepatitis. Although the electrophoretic patterns of polypeptide expression were very similar with respect to the overall spot patterns, a number of polypeptides which differed either qualitatively or quantitatively were noted. Two constitutively expressed cytosolic polypeptides, P29.5 (Mr 29.5 kDa/pI 6.73) and P30 (30 kDa/6.70), were not detected in livers of LEC animals at any age. In the normal LEA rats both P29.5 and P30 were detected as early as one day after birth and both were expressed at similar concentrations at all ages. In the LEC rats P30-C (30 kDa/6.68) was constitutively expressed in close proximity to the expected position of P30, and P30-C was not detected in the LEA rats. By means of non-equilibrium pH gradient electrophoresis two relatively basic polypeptides were detected in the LEC rats. P18ne was detected immediately prior to and P27ne immediately after the clinical manifestation of hepatitis. Experiments in F1 backcross ([LEA x LEC] x LEC) animals, however, failed to demonstrate any genetic link between either the expression or lack of expression of P29.5, P30, P30-C, or P18ne and hepatitis development. P27ne was detected in all backcross animals exhibiting hepatitis, but was never observed in LEC rats prior to the onset of hepatitis. Although we were unable to identify any unique loss of expression of polypeptides which are genetically linked to hepatitis susceptibility in LEC rats, specific subsets of quantitatively modulated polypeptides were detected.