OBJECTIVETo investigate the changes of oval cell proliferation rate in the rat 2-acetylaminofluorene/partial hepatectomy (2-AAF/PH) model.

METHODSLivers were collected from 2-AAF/PH rats at different time points after hepatectomy. Paraffin sections were investigated by double immunofluorescent staining with confocal microscopy for oval cell marker epithelial cell adhesion molecule and proliferative index proliferating cell nuclear antigen, or epithelial cell adhesion molecule and alpha-smooth muscle actin. Deposition of matrix in liver tissue was detected by sirius red staining.

RESULTSResponse of ductular oval cells could be observed in portal area at 2 days after PH, and the number of oval cells reached its peak at 9 days and then gradually declined. Oval cell proliferation rate decreased from (91.3 +/- 1.6)% at 2 days after PH to (53.6 +/- 4.4)% at 12 days (P < 0.01). In addition, oval cells infiltrating into liver parenchyma were closely associated with activated hepatic stellate cells and extracellular matrix.

CONCLUSIONSOval cell proliferation rate starts decreasing before its number reaches a peak in 2-AAF/PH model. Hepatic stellate cells probably tightly regulate oval cell number through secreting several factors and producing extracellular matrix.

2-Acetylaminofluorene ; pharmacology ; Animals ; Cell Division ; Cell Proliferation ; Hepatectomy ; Liver ; cytology ; Male ; Rats ; Rats, Sprague-Dawley ; Stem Cells ; cytology

Objective: To investigate the dynamic expression pattern of carcinoembryonic Wnt3a and its early monitoring value using a hepatocellular carcinoma model. Methods: Forty-eight Sprague Dawley (SD) rats were fed with pellet feed containing 2-acetylaminofluorene (2-AAF, 0.05%) to induce hepatocarcinogenesis, and control rats were fed a pellet diet. Liver tissue and blood samples were collected every two weeks. Liver tissues were pathologically examined using HE staining and grouped. The gene and Wnt3a mRNA expression were analyzed by genome-wide microarray. The expression and distribution of Wnt3a in liver tissue were analyzed by immunohistochemistry. Wnt3a concentration in liver tissue and serum was quantified by enzyme-linked immunosorbent assay. Statistical methods such as χ² test, Mann-Whitney test and analysis of variance were used to analyze the differences between groups. Results: According to the pathological examination results, the rat livers were divided into four groups: control, hepatocyte degeneration, precancerous lesions and hepatocellular carcinoma. Genome-wide expression profiling analysis and comparison with the control group revealed that 268 and 312 genes were up-regulated and 57 and 201 genes were down-regulated in the precancerous and cancerous group when signal logarithm ratio (SLR) was >8 log₂^cy5/cy3, and these significantly altered genes mainly involved in cell proliferation, signal transduction, tumor metastasis, and apoptosis. The expression of Wnt3a at mRNA level was significantly increased in all stages of cancer induction, including degeneration group (1.15±0.24, q=8.227), precancerous group (1.85±0.18, q=12.361) and cancerous group (2.59±0.55, q=18.082). Compared with the control group (0.25±0.11, F=121.103, P<0.001), the degeneration group, the precancerous group and the liver cancer group were up-regulated by 4.6, 7.4 and 10.4-folds, respectively. Immunohistochemistry showed that compared with the control group, the positive rate of Wnt3a in the degeneration group was 66.7% (12/18, χ²=10.701, P=0.001), and both the precancerous and liver cancer groups were positive (9/9, χ²=17.115, P<0.001). Wnt3a expression was gradually increased in liver and blood samples during the process of carcinogenesis, and the difference between two groups was statistically significant (F=176.711, P<0.001). Wnt3a overexpression was secreted into blood stream via cancerous liver tissue, and there was a linear correlation between Wnt3a levels in blood and liver samples (r=0.732, P<0.001). Conclusions: Wnt3a overexpression is closely related with hepatocellular carcinogenesis, and thus may become a new monitoring marker.
Rats ; Animals ; Carcinoma, Hepatocellular/pathology* ; Liver Neoplasms/pathology* ; Rats, Sprague-Dawley ; Carcinogenesis/metabolism* ; 2-Acetylaminofluorene ; RNA, Messenger/metabolism* ; Precancerous Conditions

Three different chemical carcinogens, 2-acetylaminofluorene (AAF), diethylnitrosamine(DENA), and 3'-methyl-4dimethylaminoazobenzene (3'-Me DAB) were used to induce hepatomas in rats. Plasma membrane surface proteins of normal rat liver cells and rat hepatomas were extracted with 3M KCI. From the analysis of the proteins of normal rat liver and rat hepatoma induced by 3'-Me DAB by discontinuous polyacrylamide gel electrophoresis(Disc-PAGE), under nonreducing and nondenaturing conditions polyacrylamide gel electrophoresis in the presence of SDS and 2-mercaptoethanol (SDS-PAGE), Sephadex G-200 gel permeation chromatography, DEAE-A50 ion-exchange chromatography and two-dimensional gel electrophoresis, at least three tumor specific antigens were identified. One had a molecular weigh of 66,000 (pl=6.79) while the other two had the same molecular weight 73,000 but differed in their isoelectric points (7.58 and 7.81). For immunological analysis of tumor specific antigens, the absorbed antiserum was prepared. Plasma membrane surface proteins of rat hepatoma induced by 3'-Me DAB were used to obtain New Zealand White male rabbit antiserum. Rabbit antiserum was then reacted with the proteins isolated from the plasma membrane surface of normal rat liver and the absorbed antiserum reacting specifically with the tumor specific antigens derived by 3'-Me DAB was obtained. Using the absorbed antiserum, the immunoreactivities of plasma membrane surface proteins isolated from rat hepatomas induced by 3'-Me DAB, AAF, and DENA were compared by Ouchterlony double immunodiffusion analysis and immunoelectrophoresis. To characterize the proteins reacting to the absorbed antiserum, immunoglobulin G was separated from the absorbed antiserum and coupled to cyanogen bromide-activated Sepharose CL-4B. The isolated proteins from the plasma membrane surface proteins of 3'-Me DAB-induced hepatoma using this immunoaffinity chromatography had molecular weights of 66,000 and 73,000. The localization of these proteins on surface plasma membranes of rat hepatomas induced by 3'-Me DAB was confirmed by an immunofluorescence technique. The experimental results revealed the existence of cross-reacting common antigens on the plasma membrane surface of rat hepatomas induced by different hepatocarcinogens.
2-Acetylaminofluorene ; Animal ; Antigens, Neoplasm/*isolation and purification ; Antigens, Surface/isolation and purification ; Diethylnitrosamine ; Liver Neoplasms, Experimental/chemically induced/*immunology ; Methyldimethylaminoazobenzene ; Rats ; Rats, Inbred Strains ; Support, Non-U.S. Gov't

In summarizing the results of the experimental studies up to the present, it is conjectured that the pathogenicity of Entamoeba histolytica or establishment of amoebiasis is not unique but differs by strain and age of Entamoeba histolytica and the age of the host. A non-virulent strain is more likely adapt to as low a temperature as 32 . This is not so in the strains which originated form clinical cases. Iso-enzyme patterns may roughly characterize pathogenic strains from non-pathogenic, Red blood cells may contribute as nutrients for growth of Entamoeba histolytica only after they have been hemolysed, but they are toxic to the amoebae as long as they remains intact. A low protein diet and stress may facilitate the establishment of amoebiasis; male sex hormones or previous infection by enteric bacteria provide a more advantageous condition than the female; and hepatotoxic agents will accelerate amoebic hepatitis.
2-Acetylaminofluorene ; Animal ; Antigens, Neoplasm/*isolation and purification ; Antigens, Surface/isolation and purification ; Diethylnitrosamine ; Liver Neoplasms, Experimental/chemically induced/*immunology ; Methyldimethylaminoazobenzene ; Rats ; Rats, Inbred Strains ; Support, Non-U.S. Gov't

The purpose of this study was to investigate the effects of green tea ingestion on hepatocarcinogenesis before and after its initiation. Male Sprague-Dawley rats were fed an AIN76A diet with or without green tea. Initiation was induced by a single dose (200 mg/kg) of diethylnitrosamine at week 4 and 0.02% (w/w) 2-acetylaminofluorene was supplied in the diets. The control group had free access to water for 13 weeks (CTR13). Tea infusion was provided from the beginning of the experiment for 13 weeks (PRE13) or from the post-initiation stage until week 13 (POST13). Three other groups (CTR24, PRE24 and POST24) were added to examine the longer-term effects (24 weeks) with the same experimental design. The percentage area of liver sections that were positive for hepatic placental glutathione S-transferase (GST-P), which was used as a marker of preneoplastic lesions, was smaller in PRE13 (20.2 +/- 5.0%, mean +/- SD) and POST13 (26.0 +/- 4.8%) than in CTR13 (33.2 +/- 5.8%, p<0.05). Over the longer period, the GST-P lesions were significantly smaller for both PRE24 and POST24 (21.6 +/- 8.5% and 22.2 +/- 4.0%, respectively) than for CTR24 (28.6 +/- 5.1%, p<0.05), but there was no significant difference between PRE24 and POST24. The liver content of thiobarbituric acid reactive substances was significantly lower in the tea groups than in the controls (p<0.05). However, no significant differences were observed among groups of GST activity. The results show that tea consumption exhibits a stronger short-term initiation-inhibiting ability in liver carcinogenesis, but over a longer period, the preventive effects of green tea ingestion do not differ in post- and pre-initiation.
2-Acetylaminofluorene ; Animals ; Diet ; Diethylnitrosamine ; Eating ; Glutathione Transferase ; Humans ; Liver ; Male ; Rats ; Rats, Sprague-Dawley ; Research Design ; Tea ; Thiobarbiturates ; Thiobarbituric Acid Reactive Substances ; Water

PURPOSE: The proliferative activity of cells in enzyme altered fdegrees Ci of the rat hepatoma model was measured by double immunohistdegrees Chemical staining methods using anti-bromodeoxyuridine (BrdU) and anti-glutathione S transferase of placental form (GST-P). The aim of this study was to compare the cell proliferative activity in GST-P positive altered fdegrees Ci and in negative fdegrees Ci. MATERIALS AND METHODS: Eight-week-old male Sprague-Dawley rats were administered by 200 mg/kg diethylnitrosamine (DEN) intraperitoneally, and followed by 0.02% acetylaminofluorene (AAF)-containing diet for 4 weeks. One week after administration of AAF diet, two-thirds hepa tectomy was performed. Control animals were treated as same except for the omission of AAF in the diet. The rats were sacrified 0, 1, 3, 5, 7, 14 and 21 days after partial hepatectomy. The slices of liver were fixed in acetone, dehydrated in benzene and stained by peroxidase-anti peroxidase method against GST-P and by avidine-biotin peroxidase complex method against BrdU. RESULTS: The area of the GST-P positive fdegrees Ci was increased during the experimental period. In the experimental group, the S-phase fraction in the fdegrees Ci remained high during the first week and was decreased thereafter. However, the GST-P negative area maintained a low S-phase cell frac tion throughout the experimental period. CONCLUSION: These results suggest that hepatic cells in the enzyme altered fdegrees Ci may escape a suppressor effect of AAF in contrast to the normal cells in which their growth are inhibited by AAF.
2-Acetylaminofluorene ; Acetone ; Animals ; Benzene ; Bromodeoxyuridine ; Carcinogenesis* ; Carcinoma, Hepatocellular ; Diet ; Diethylnitrosamine ; Hepatectomy ; Hepatocytes ; Humans ; Liver ; Male ; Peroxidase ; Rats* ; Rats, Sprague-Dawley ; Transferases ; United Nations

We observe changes of activation of hepatic microsomal cytochrome p-450, changes of formation of riug-and N-hydroxylation of 2-acetylaminofluorene, and changes of vitamin D, in the liver which exposed to UV light and expoeed to UV light after being applied by sunscreens. The results of our study are as shown below: 1. The contents of hepatic microsomal cytochrome p-45p of newborn rats were found to be remarkably increase in the group exposed to UV light for 3 weeks (p<0. 001), but such changes were much reduced in the group exposed to UV light for 3 weeks after being applied by sunscreens(p<0.05. p<0.001, ) 2.Cytochrome p-450 induced by UV light was found to be significantly increased ring and N-hydroxylation of 2-acetylarninofluorene known as carcinogenic source for the liver. In the group exposed to UV light for 3 weeks after being applied by sunscreens, both of ring-and N-hydroxylation of 2-acetylaminofluorene were significantly reduced(p<0. 0, p<0. 001). 3, The contents of vitarnin D, in the liver of newborn rats were found to be gradually increased when they were exposed to UV light for 1 week or 3 weeks (p<0. 001), and in the group exposed to UV light after being applied by sunscreens, such changes were reduced remarkably(pg0. (301).
2-Acetylaminofluorene ; Animals ; Cholecalciferol ; Cytochrome P-450 Enzyme System ; Cytochromes* ; Humans ; Infant, Newborn* ; Liver ; Rats* ; Sunscreening Agents* ; Ultraviolet Rays ; Vitamin D* ; Vitamins*

OBJECTIVETo investigate the possible mechanisms of miR-21-mediated regulation of proliferation and activation of hepatic oval cells.

METHODSThe 2-acetamidofluorene/partial hepatectomy (2-AAF/PH) method was applied to generate hepatic oval cell activation model in male Sprague-Dawley rats; after the 7 days of 2-AAF/PH or PH alone (control), the rats were sacrificed at 0 h, 6 h, 12 h, 24 h, 72 h and 168 h. Expression of miR-21 was detected by real-time PCR and differences between groups were evaluated using the two-sample t-test. Differential transcription of miR-21 target genes was assessed bioinformatically, and with western blotting to detect changes in protein expression of the target gene.

RESULTSThe rat hepatic oval cell activation model was successfully established.The 2-AAF/PH rats showed miR-21 expression beginning to increase at 12 h, peaking at 24 h, and decreasing thereafter until an increase at 168 h.For the control group, the miR-21 expression began to increase at 6 h, until 24 h when expression began steadily declining to reach the original level.Compared to the control group, the experimental group showed expression of miR-21 that was significantly less at 6 h (P=0.039, t =3.029) and significantly more at 24 h and 168 h (P=0.026, t =-3.433 and P=0.007, t =-5.105). Among the predicted target genes of miR-21 were WW domain containing E3 ubiquitin protein ligase 1 (WWD), Smad family member 7 (Smad7), and polybromo-1 (Pbrm1).Smad7 protein expression began to decrease at 6 h in the control group, until reaching its minimum at 24 h when it increased; in the experimental group, SMAD7 expression increased at 6 h, then began to decrease with the minimum detected at 168 hour.In the control group, the Smad7 mRNA expression decreased slightly at 6 h, then began to increase, reaching its peak at 24 h when the expression fell to the original level. In the experimental group, the Smad7 mRNA expression began to increase at 6 h and reached its peak at 24 h when it decreased; the expression was little more than its original level at 168 h.Smad7 protein expression was negatively correlated with miR-21, and Smad7 mRNA expression was positively correlated with miR-21 but negatively correlated with Smad7 protein expression.

CONCLUSIONmiR-21 may play a vital role in the activation and proliferation of hepatic oval cells.As a target gene of miR-21, Smad7 might be involved in the process.

2-Acetylaminofluorene ; Animals ; Cell Proliferation ; Hepatectomy ; Hepatocytes ; cytology ; Liver ; cytology ; Male ; MicroRNAs ; genetics ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction

BACKGROUND/AIMS: In the 2-acetylaminofluorene (2-AAF)/70% partial hepatectomy (PHx) model, the mechanism underlying the differentiation of activated hepatic oval cells (HOCs) into hepatocytes and bile ductile cells is unclear. We investigated the role of cyclooxygenase-2 (COX-2) in HOCs and the relationship between COX-2 and extracellular matrix proteins in cellular proliferation. METHODS: Reverse transcription-polymerase chain reaction, immunohistochemical staining, and Western blotting were used to assess COX-2 expression. The co-localization of COX-2 with Thy1, c-Met, epithelial cell adhesion molecule, and alpha-smooth muscle actin was also examined. Additionally, we investigated whether connective tissue growth factor (CTGF), fibronectin (FN), extracellular signal-regulated kinase 1/2 (P-ERK1/2), and AKT were expressed in HOCs. RESULTS: The expression of COX-2, prostaglandin E2 receptors, and c-Met was upregulated in HOCs. However, HOCs treated with the COX-2 inhibitor NS398 showed decreased COX-2, CTGF, FN, and AKT expression, whereas P-ERK1/2 was unaffected. Additionally, NS398 inhibited HOC proliferation, but not the proliferation of HOCs cultured on FN-coated dishes. Furthermore, the proliferative response of HOCs treated with NS398 was reversed by hepatic growth factor treatment. CONCLUSIONS: These results suggest that HOC proliferation is mediated through COX-2, extracellular FN expression, and AKT activation. Thus, COX-2 plays an important role in HOC proliferation following acute injury.
2-Acetylaminofluorene ; Actins ; Animals ; Antigens, Neoplasm ; Bile ; Blotting, Western ; Cell Adhesion Molecules ; Connective Tissue Growth Factor ; Cyclooxygenase 2 ; Dinoprostone ; Epithelial Cells ; Extracellular Matrix ; Extracellular Matrix Proteins ; Fibronectins ; Hepatectomy ; Hepatocytes ; Liver ; Liver Regeneration ; Muscles ; Nitrobenzenes ; Phosphotransferases ; Rats ; Sulfonamides

Administering of 2-acetylaminofluorene (2-AAF) before a two-thirds partial hepatectomy (PHx) results in suppression of hepatocyte proliferation and stimulation of oval cell proliferation. The objectives of this study was to examine the oval cell behaviour and associated transforming growth factor-beta1 (TGF-beta1) protein expression by combining 2-AAF with selective hepatic damage caused by PHx. We also studied the temporal relationship between TGF-beta1 expression, and proliferation and apoptosis of oval cells. Oval cells emerged from the portal areas and became more numerous with time fanning out into the periportal and midzonal hepatic parenchyma. Both smooth muscle actin (SMA) and TGF-beta1 immunostain revealed that TGF-beta1-positive cells were SMA-positive hepatic stellate cells (HSCs). Coinciding with the proliferation of oval cells, an increase expression of TGF-beta1 produced by SMA-positive HSCs was observed, thereafter apoptosis of oval cells reached its peak. This result implicated that TGF-beta1 produced by HSCs is intimately associated with proliferation and apoptosis of oval cells, and plays a role in the cessation of oval cell activation and remodeling of liver parenchyma in 2-AAF induced liver regeneration.
2-Acetylaminofluorene ; Animal ; Apoptosis/physiology* ; Hepatectomy ; Immunohistochemistry ; In Situ Nick-End Labeling ; Liver/ultrastructure ; Liver/metabolism* ; Liver/cytology ; Liver Regeneration/physiology* ; Liver Regeneration/drug effects ; Male ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta/metabolism*