The present study was designed in order to investigate the lectin induced cytoagglutination properties of normal and transformed cells and surface alterations in the early stage of the transformed cells by characterizing the structural changes on the hepatoma surface membrane. Rat and rabbit erythrocytes and Sarcoma 180 ascites tumor cells were used for the lectin-induced cytoagglutination. Plotting % agglutination versus concanavalin A(Con A) concentration, sigmoid curves appeared in all cases. alpha-methyl-D-mannopyranoside(alphaMM) inhibited Con A induced cytoagglutination and the degrees of inhibition depended on the cell types and species. When rats were fed a diet containing 0.06% 3'-methyl-4dimethylaminoazobenzene(3'-Me DAB) for 12 weeks, almost all of the rats had solid liver tumors. Polyacrylamide gel electrophoresis of surface membrane proteins of these rat livers and of transplanted tumor cells showed three distinct protein bands, of which two were absent in normal rat livers. The molecular weights of these proteins were 73,000, 66,000, and 57,000 daltons. Antiserum against primary hepatocarcinoma surface proteins precipitated with three membrane proteins obtained from primary hepatocarcinoma cells as well as transplanted hepatocarcinoma cells, suggesting the presence of specific tumor antigens in these cells.
Animal ; Cell Membrane/pathology* ; Cell Transformation, Neoplastic/ultrastructure* ; Concanavalin A/diagnostic use ; Liver Neoplasms, Experimental/chemically induced ; Liver Neoplasms, Experimental/ultrastructure* ; Methyldimethylaminoazobenzene ; Rabbits ; Rats ; Surface Properties

OBJECTIVETo investigate the role of the spleen on hepatocyte proliferative activity during experimental hepatocarcinogenesis in rats.

METHODSCell-cycle and expression of proliferating cell nuclear antigen (PCNA) of hepatocytes were studied by immunohistochemical and flow cytometric technique in two groups: splenectomy (45 rats) and sham-operation with laparotomy (45 rats).

RESULTS(1) Hepatocirrhosis was formed in group A earlier than in group B. Marked degenerative changes of the liver parachyma showing vacuolization of hepatocytes were observed in hepatic lobules. (2) The proliferative level of hepatocytes increased with the progression of hepatocarcinogenesis, and topped at the 18 th week. (3) The proliferative level of the splenectomy group was lower than that of the sham-operation group in the mid-stage of carcinogenesis (t = 4.76, P < 0.05). After stopping feeding diethylnitrosamine (DENA), however, no difference was found in the hepatocyte proliferative activity between the two groups at the 18th week.

CONCLUSIONSThere is a close relationship between hepatic proliferative activity and spleen, and the spleen may play an important role in facilitating hepatic proliferation.

Animals ; Cell Cycle ; Diethylnitrosamine ; Liver ; pathology ; Liver Neoplasms, Experimental ; chemically induced ; pathology ; Male ; Proliferating Cell Nuclear Antigen ; analysis ; Rats ; Rats, Wistar ; Spleen ; physiology ; Splenectomy

2-Acetylaminofluorene ; Angiogenesis Inhibitors ; pharmacology ; Animals ; Carcinoma, Hepatocellular ; chemically induced ; metabolism ; pathology ; Immunohistochemistry ; Liver ; drug effects ; metabolism ; pathology ; Liver Neoplasms, Experimental ; chemically induced ; metabolism ; pathology ; Male ; NF-kappa B ; antagonists & inhibitors ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Thalidomide ; pharmacology

Anticarcinogenic effect of red ginseng (Panax ginseng C.A. Meyer cultivated in JiLin, China) on the development of liver cancer induced by diethylnitrosamine (DEN) in rats was studied, especially in preventive and curative groups. In the preventive group, the rats were given with DEN concomitantly with red ginseng fluid, and in the curative group, the rats were administered with red ginseng fluid after they developed liver cancer nodules induced by DEN. The result of the preventive group revealed that the developmental rate of liver cancer in the experimental group was 14.3%, while 100% in the control group, with the difference being statistically significant. DNA, RNA, glycogen, gamma-GT, SDH, and 5'-NT were maintained at relatively normal level in experimental group, and decreased or increased in the control group. The result of curative group showed that hepatoma nodules of the DEN-red ginseng group I were smaller than those of control group I, the structure of hepatic tissue was well preserved, the area with gamma-GT positive was smaller, and the ultrastructure of hepatocytes was normal. The average life span the DEN-red ginseng group II and the DEN control group II were 72.8 and 42.3 days, respectively. To sum up, all findings on preventive and curative groups had clearly proved that the red ginseng had the anticarcinogenic effect on the development of liver cancer induced by DEN in rats.
Animal ; Anticarcinogenic Agents/*pharmacology ; Carcinoma, Hepatocellular/chemically induced/*pathology ; Data Interpretation, Statistical ; Diethylnitrosamine/adverse effects ; Liver/pathology/ultrastructure ; Liver Neoplasms, Experimental/chemically induced/*pathology ; Male ; *Panax ; Plant Extracts/pharmacology ; Rats ; Rats, Wistar

To investigate the influence of bear bile on rat hepatocarcinoma induced by diethylnitrosamine (DEN), a total of 40 rats were randomly divided into 4 groups: normal control group, model group, and two bear bile treatment groups. The rat liver cancer model was induced by breeding with water containing 100 mg x L(-1) DEN for 14 weeks. The rats of the bear bile groups received bear bile powder (200 or 400 mg x kg(-1)) orally 5 times per week for 18 weeks. The general condition and the body weight of rats were examined every day. After 18 weeks the activities of serum alanine transaminase (ALT), aspartate transaminase (AST) and total bilirubin (TBIL) were detected. Meanwhile, the pathological changes of liver tissues were observed after H&E staining. The expression of proliferative cell nuclear antigen (PCNA) and a-smooth muscle actin (alpha-SMA) in liver tissue were detected by immunohistochemical method. After 4 weeks the body weights of rats in normal group were significantly more than that in other groups (P < 0.05); and that in the two bile groups was significantly more than that in the model group. Compared with normal group, the level of serum glutamic-pyruvic transaminase and total bilirubin increased significantly in other groups; compared with model group, these two indexes decreased significantly in two bile groups. Hepatocellular carcinoma occurred in all rats except for normal group; there were classic cirrhosis and cancer in model group while there were mild cirrhosis and high differentiation in two bile groups. There were almost no expressions of PCNA and alpha-SMA in normal group while there were high expressions in model group; the two bile groups had some expressions but were inferior to the model group, and alpha-SMA reduced markedly. It indicated that bear bile restrained the development of liver cancer during DEN inducing rat hepatocarcinoma, which may be related to its depressing hepatic stellate cell activation and relieving hepatic lesion and cirrhosis.
Actins ; metabolism ; Alanine Transaminase ; blood ; Animals ; Antineoplastic Agents ; pharmacology ; Aspartate Aminotransferases ; blood ; Bile ; chemistry ; Bilirubin ; blood ; Body Weight ; drug effects ; Carcinoma, Hepatocellular ; blood ; chemically induced ; pathology ; Diethylnitrosamine ; Liver ; metabolism ; pathology ; Liver Cirrhosis ; chemically induced ; pathology ; Liver Neoplasms, Experimental ; blood ; chemically induced ; pathology ; Male ; Powders ; pharmacology ; Proliferating Cell Nuclear Antigen ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Ursidae

With the aim of establishing bio-indices for the development of multistep hepatotumorigenesis, rats were fed water containing 0.01% diethylnitrosamine (DEN) ad libitum for 13 weeks. This treatment with DEN only made it possible to induce hepatic tumors in 100%. After the DEN administration, several clinical symptoms were observed including minor behavioral changes, brittleness of hair and a decrease in water and food intake. The concentration of total serum protein and albumin in all treated groups was significantly lower than in non-treated controls (p<0.05). Increase of specific enzyme (AST, ALT and GGT) activity (p<0.05), variable tumor size and hepatomegaly of the liver was observed in all rats treated with DEN for 10 weeks. Both hepatocellular carcinoma and cholangiocarcinoma were found in the same livers at the same time, and were prominently developed after 12 weeks. In case of carcinoma, some of the livers showed more or less advanced states over the 12-15 weeks period. In the present study, hepatocellular carcinoma was developed by treating DEN in only the drinking water, without any other carcinogens or without partial hepatectomy. These results indicate that DEN is a new carcinogen that acts directly on it the liver, moreover, it might be very useful for investigating hepatotumorigenesis.
Alanine Transaminase/blood ; Animals ; Aspartate Aminotransferases/blood ; Biological Markers/blood ; Carcinogens ; *Cell Transformation, Neoplastic ; Diethylnitrosamine/toxicity ; Liver/drug effects/*pathology ; Liver Neoplasms/blood/chemically induced/*pathology ; Liver Neoplasms, Experimental/blood/*pathology ; Male ; Rats ; Rats, Sprague-Dawley ; gamma-Glutamyltransferase/blood

BACKGROUNDIt is generally accepted that spleen plays a complex role in the tumor immunity, which would change in the different periods of cancer. In this study, we investigated the changes in the function of splenic macrophage (Mphi) in different stages of liver cancer induced by diethylnitrosamine (DEN) in rats. The aim was to support the characteristics of "two-way" and "phase" of spleen in tumor immunity.

METHODSThe model of pulmonary metastasis of liver cancer was established in forty male SD rats by DEN. In the 8th, 13th and 16th week, 10 rats were randomly chosen and sacrificed, and divided into cirrhosis, liver cancer and pulmonary metastasis groups depending on the pathological result, respectively. The other 10 rats were taken as control group. The Mphi was isolated by anchoring cultivation. The changes in ultrastructure, phagocytosis, cytokine secretion, antigen processing and presenting, and viability of splenic Mphi were detected by transmission electron microscopy, Vybrant(TM) Phagocytosis Assay, DQ(TM) Ovalbumin, and rat TNF-alpha ELISpot kits.

RESULTSUnder the electron microscope, the Mphi in the control group had some pseudopodium-like prominences, and mitochondria, ribosome, rough endoplasmic reticulum, lysosome can be found in the cytoplasm, and phagocytized RBC. In the liver cirrhosis and liver cancer group, Mphi had more prominences, meanwhile much more mitochondria, ribosome, rough endoplasmic reticulum, lysosome can be found in the cytoplasm, especially in the liver cancer group. In the pulmonary metastasis group, the Mphi was swelling, with few organelle. As compared to the control group, the function of splenic Mphi increased in cirrhosis and cancer groups, but decreased in metastasis group (phagocytosis rate: (84.7 +/- 1.9)%, (89.5 +/- 3.1)%, and (36.0 +/- 2.6)% vs (75.6 +/- 1.7)%, P < 0.05, P < 0.01; viability: (1.53 +/- 0.15)%, (1. +/- 0.14)%, and (1.12 +/- 0.29)% vs (1.48 +/- 0.17)%, P < 0.05, P < 0.01; TNF-alpha secretion: (741.0 +/- 52.9)%, (1126.2 +/- 174.5)%, and (313.8 +/- 50.8)% vs (626.6 +/- 24.6)%, P < 0.05, P < 0.01; positive cell rate of antigen processing and presenting: (24.03 +/- 1.87)%, (27.95 +/- 2.63)%, and (10.46 +/- 2.16)% vs (16.45 +/- 1.86)%, P < 0.01).

CONCLUSIONSIn the stage of cirrhosis and early cancer, the immune functions of splenic Mphi were reinforced. It may promote the non-specificity tumor immunity. On opposite, in the stage of pulmonary metastasis, the immune functions of splenic Mphi were impaired. It may lead to the decrease of tumor immunity.

Animals ; Cells, Cultured ; Diethylnitrosamine ; toxicity ; Disease Models, Animal ; Liver Cirrhosis ; immunology ; pathology ; Liver Neoplasms, Experimental ; chemically induced ; complications ; immunology ; ultrastructure ; Lung Neoplasms ; immunology ; secondary ; ultrastructure ; Macrophages ; pathology ; ultrastructure ; Male ; Microscopy, Electron, Transmission ; Rats ; Rats, Sprague-Dawley ; Spleen ; pathology ; ultrastructure

BACKGROUND/AIMS: ErbB receptor proteins are transmembrane tyrosine kinase receptors; when they are activated by interaction with ligands, they generate diverse cellular responses, especially during lesion development and progression to cancer. In this study the expression of ErbB receptors and TGF-alpha were investigated using an experimental cirrhosis rat model giving rise to hepatocellular neoplasms, similar to human liver diseases. METHODS: Fifty three male rats received intraperitoneal injection of diethylnitrosamine (DEN, 50 mg/kg), weekly for 18 weeks. Until the eighth week, two rats were sacrificed every two weeks and from the tenth to the eighteenth week, five rats were sacrificed weekly. Grossly, dyschromatic and dysmorphic nodules were counted and categorized into three groups: N1/N2/N3: 3 mm < or = x < 5 mm/5 mm < or = x < 10 mm/x > or = 10 mm in diameter. All nodules were examined, histologically. Antibodies for GSTp, TGF-alpha, EGF-R, ErbB2, ErbB3 and ErbB4 were used for immunohistochemistry. RESULTS: The onset of cirrhoses was noted from the twelfth week. Preneoplastic foci, hepatocellular adenomas (HCA) and hepatocellular carcinomas (HCC) were noted from the second, eleventh and fifteenth week, respectively. The nodules (N1/N2/N3: 397/258/64) included regenerating nodule; RN (N1/N2/N3: 72.3%/15.9%/0%), HCA (N1/N2/N3: 27.2%/82.2%/7.6%) and HCC (N1/N2/N3: 0.5%/ 1.9%/92.4%). EGF-R was expressed in 12.5% of RN, 64.7% HCA and 75.2% HCC. TGF-alpha was expressed in 92.4% of RN, 91.3% HCA and 93.2% HCC. Sixty eight percent of TGF-alpha expressing nodules showed concurrent EGF-R expression. ErbB2 was expressed in 83.6% of RN, 72.9% HCA and 88.7% HCC. ErbB4 was expressed in 95.2% of RN, 86.3% HCA and 62.5% HCC. CONCLUSIONS: Increased expression of EGF-R and decreased expression of ErbB4, might be related with tumor progression during DEN-induced hepatocarcinogenesis.
Adenoma, Liver Cell/chemically induced/metabolism/pathology ; Animals ; Carcinoma, Hepatocellular/chemically induced/*metabolism/pathology ; Diethylnitrosamine ; Glutathione Transferase/metabolism ; Immunohistochemistry ; Liver Neoplasms, Experimental/chemically induced/*metabolism/pathology ; Male ; Rats ; Rats, Wistar ; Receptor, Epidermal Growth Factor/*metabolism ; Receptor, erbB-2/*metabolism ; Transforming Growth Factor alpha/*metabolism

To study the effects of Smad4 on liver fibrosis and hepatocarcinogenesis in mice treated with CCl(4)/ethanol. The wild-type mice (Smad4 +/+) and the Smad4 knockout mice (Smad4 +/-) were injected subcutaneously with carbon tetrachloride(CCl(4))/ethanol twice a week for twenty weeks. The expression of Smad4, TGFbeta1, Smad2, Smad3, Smad6, TIMP1, MMP2 and MMP9 was detected by RT-PCR. In the cirrhotic liver, the expression of Smad4 mRNA was significantly higher than that in the normal liver. Comparing with wild-type mice (Smad4 +/+), the TGFbeta1-Smad4 signaling was markedly attenuated in the Smad4 knockout mice (Smad4 +/-). After induction by CCl(4)/ethanol, the hepatic fibrosis in the Smad4 knockout mice (Smad4 +/-) was obviously alleviated compared with the wild-type mice (Smad4 +/+), and the incidence rate of hepatocarcinogenesis of the former was also lower than that of the latter(32.0% vs 41.9%). These results indicate that knocking out Smad4 can delay the progression of liver fibrosis and liver cancer.
Animals ; Carbon Tetrachloride ; administration & dosage ; Disease Models, Animal ; Ethanol ; administration & dosage ; Female ; Liver Cirrhosis, Experimental ; chemically induced ; metabolism ; pathology ; Liver Neoplasms, Experimental ; chemically induced ; metabolism ; pathology ; Male ; Mice ; Mice, Knockout ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; Smad Proteins ; genetics ; metabolism ; Smad4 Protein ; genetics ; metabolism ; Tissue Inhibitor of Metalloproteinase-1 ; genetics ; metabolism ; Transforming Growth Factor beta1 ; genetics ; metabolism

OBJECTIVETo investigate the effects of herbal compound 861 (Cpd861) on hepatocarcinogenesis induced by diethylntrosamine and 2-acetylaminofluorene (DEN-AAF) in female Sprague Dawley rats.

METHODSLiver preneoplastic foci were induced using the DEN-AAF method in female Sprague Dawley rats, which were then treated with Cpd861. For quantitative assessment of liver preneoplastic foci, the placental form of glutathione-S-transferase (GST-P) positive foci were measured using immunohistochemical staining and image analysis. GST-P protein expression was measured by Western blotting, mRNA expression was assessed by Northern blotting.

RESULTSTreatment using DEN-AAF caused a significant decrease in body weight and increase in liver weight compared to the control group. Oral Cpd861 administration essentially prevented DEN-AAF-induced body weight loss and liver weight increase. When 2-AAF was followed by treatment with Cpd861, there was a decrease in the number of large foci as compared to 2-AAF alone. However, there were still considerable numbers of small mixed clear/vacuolated cell foci, some of which were positive for GST-P. Significant increase in GST-P protein and mRNA expression were observed in the DEN-AAF group, while treatment with Cpd861 inhibited the increase. The effect of Cpd861 on hepatocarcinogenesis occurred in a concentration-dependent manner.

CONCLUSIONChinese herbal compound Cpd861 prevents hepatocarcinogenesis in DEN-AAF-induced liver preneoplastic lesions in rats.

2-Acetylaminofluorene ; Animals ; Blotting, Northern ; Blotting, Western ; Body Weight ; Diethylnitrosamine ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Glutathione Transferase ; genetics ; Liver ; pathology ; Liver Neoplasms, Experimental ; chemically induced ; drug therapy ; pathology ; Organ Size ; Rats ; Rats, Sprague-Dawley