Ethanol extract of Phellinus merrillii protects against diethylnitrosamine- and 2-acetylaminofluorene-induced hepatocarcinogenesis in rats / 中国结合医学杂志

<p><b>OBJECTIVE</b>To study whether the ethanol extract of Phellinus merrillii (EPM) has chemopreventive potential against liver carcinogenesis.</p><p><b>METHODS</b>Thirty male Spraque-Dawley rats were randomly divided into control group, EPM control group, hepatocarcinoma control group, low-dose EPM group and high-dose EPM group, 6 in each group. Using the Solt and Farber protocol in a rat model of hepatocarcinogenesis, the chemopreventive effect of EPM on diethylnitrosamine (DEN)-initiated, 2-acetylaminofluorene (2-AAF) and partial hepatectomy (PH)-promoted liver carcinogenesis in rats was evaluated. Basic pathophysiological and histological examinations, together with the serum levels of glutamic oxaloacetic transaminase (sGOT), glutamic pyruvic transaminase (sGPT) and gamma-glutamyl transpeptidase (γ-GT) were measured.</p><p><b>RESULTS</b>Treatment of EPM at the concentration of 2 g/kg body weight in the diet for 8 weeks clearly prevented the development of carcinogenesis and reduced the levels of sGOT, sGPT, and serum γ-GT of rats as compared with the hepatocarcinoma control group (P<0.05 or P<0.01). These phenotypes were accompanied by a significant increase in natural killer cell activity.</p><p><b>CONCLUSION</b>EPM showed a strong liver preventive effect against DEN+2-AAF+PH-induced hepatocarcinogenesis in a rat model.</p>

Role of miR-21 in rat hepatic oval cell proliferation and activation / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the possible mechanisms of miR-21-mediated regulation of proliferation and activation of hepatic oval cells.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>miR-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.</p>

Proliferation of Hepatic Oval Cells via Cyclooxygenase-2 and Extracellular Matrix Protein Signaling during Liver Regeneration Following 2-AAF/Partial Hepatectomy in Rats

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.

A study of oval cell proliferation kinetics in the rat 2-acetylaminofluorene/partial hepatectomy model / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the changes of oval cell proliferation rate in the rat 2-acetylaminofluorene/partial hepatectomy (2-AAF/PH) model.</p><p><b>METHODS</b>Livers 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.</p><p><b>RESULTS</b>Response 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.</p><p><b>CONCLUSIONS</b>Oval 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.</p>

protective effects of demethoxyviridine and 1-alpha-hydroxy-demethoxyviridine in the livers of male rats treated with diethylnitrosamine and 2-acetylaminflourene

To determine the protective effects of a fungal metabolite of demethoxyviridine [DMV] and its derivative, 1-alpha-hydroxy-DMV in the livers of 2-month-old male Spraque-Dawley rats treated with diethylnitrosamine [DEN] and 2-acetylaminflourene [2-AAF]. This study was performed in the Department of Medical Biology, Faculty of Medicine, Eskisehir Osmangazi University, Eskisehir, Turkey from May 2006. Animals were divided into 10 groups. Those were the control, olive oil, dimethyl sulfoxide [DMSO], DMV, 1-alpha-hydroxy-DMV, DEN, 2-AAF, DEN+2-AAF, DEN+2-AAF+DMV, and DEN+2-AAF+1-alpha-hydroxy-DMV-treated animal groups. The liver microsomes were prepared from rats and the levels of expression of cytochrome P450 1A2 [CYP1A2] enzymes were determined with western blot technique. The liver tissue slides were evaluated histopathologically with hematoxylin and eosin staining and immunohistochemically for Harvey-retrovirus associated DNA sequences [Ha-Ras], glutathione S- transferase [GST-p], and connexion-32 [Cx32] proteins. Notably, there were no appreciable differences in CYP1A2 level among control, olive oil, and DMSO-treated animals. The CYP1A2 level was significantly decreased in 2-AAF, DEN+2-AAF, DEN, DEN+2-AAF+DMV, DEN+2-AAF+1-alpha-hydroxy-DMV, 1-alpha-hydroxy-DMV, and DMV-treated animals as compared to the control. Most prenoplastic focus was found in DEN+2-AAF treated group. Demethoxyviridine and 1-alpha-hidroksi-DMV had protective effect in the livers of DEN, 2-AAF and DEN+2-AAF induced rats

Pre- and post-initiation modulating effects of green tea ingestion on rat hepatocarcinogenesis

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.

Chemoprevention by Butea monosperma of hepatic carcinogenesis and oxidative damage in male wistar rats.

In this communication, we document chemopreventive effects of Butea monosperma extract on hepatic carcinogenesis and on tumor promoter induced markers and oxidative stress in male Wistar rats. Treatment of male Wistar rats for five consecutive days with 2-AAF i.p. induced significant hepatic toxicity, oxidative stress and hyperproliferation. Pretreatment of B.monosperma extract (100 and 200 mg/kg body weight) prevented oxidative stress by restoring the levels of antioxidant enzymes and also prevented toxicity at both doses. The promotion parameters induced (ornithine decarboxylase activity and DNA synthesis) by 2-AAF administration in diet with partial hepatectomy (PH) were also significantly suppressed dose dependently by B. monosperma. Thereafter, we proceeded with studies on rat liver carcinogenesis. After fourteen days of DEN treatment, dietary administration of 2-AAF with PH resulted in a 100% incidence of tumors in the animals. However, B.monosperma caused reduction in the number of tumors/ rat and percentage of tumor bearing rats at the end of the study, as confirmed histologically. Thus, our data suggest that B.monosperma extract is a potent chemopreventive agent which suppresses 2-AAF-induced hepatic carcinogenesis and oxidative damage in Wistar rats. The protective activity of the plant might be due to the two major constituents (butrin and isobutrin).

Proliferation and differentiation of C-kit+ cell in vitro / 生物医学工程学杂志

This study sought to isolate and purify C-kit+ cells from rat 2-AAF/PH model and to investigate the proliferation and differentiation of C-kit+ cells in vitro. C-kit positive oval cells were enriched by using magnetic activated cell sorting (MACS). The sorted oval cells were cultured in a low density, and then colony formation was observed. The capacity of proliferation and differentiation of C-kit positive cells were examined in vitro by immunocytochemistry and RT-PCR. By using C-kit antibody in conjunction with MACS, we developed a rapid oval cell isolation protocol. The sorted cells formed colony when cultured in vitro. Cells in the colony expressed albumin or cytokeratin 19 (CK19) or coexpressed both, and BrdU incorporation test was positive. RT-PCR on colony showed expression of albumin and CK19 gene. The results demonstrate that by means of MACS we have established a method to isolate oval cells. The sorted hepatic oval cells can form colony in vitro which expresses different combinations of phenotypic markers and genes from both hepatocytes and cholangiocyte lineage.

Suppressive effect of herbal compound 861 on chemical hepatocarcinogenesis in rats / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the effects of herbal compound 861 (Cpd861) on hepatocarcinogenesis induced by diethylntrosamine and 2-acetylaminofluorene (DEN-AAF) in female Sprague Dawley rats.</p><p><b>METHODS</b>Liver 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.</p><p><b>RESULTS</b>Treatment 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.</p><p><b>CONCLUSION</b>Chinese herbal compound Cpd861 prevents hepatocarcinogenesis in DEN-AAF-induced liver preneoplastic lesions in rats.</p>

Sequential Changes of Proliferative Fraction of Enzyme Altered Fdegrees Ci in Experimental Rat Hepatdegrees Carcinogenesis / 대한암학회지

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.

Transforming growth factor-beta1 protein, proliferation and apoptosis of oval cells in acetylaminofluorene-induced rat liver regeneration

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.

Mutagenicity and anti-mutagenicity of selected spices.

Using Salmonella typhimurium strains TA 100 and TA 1535, the mutagenicity and anti-mutagenicity of extracts of several spices were checked. Spices like pepper, pippali, ginger and mustard increased the number of revertants indicating their mutagenic potential. Garlic extract on the other hand was found to inhibit the mutagenicity produced by direct acting mutagens such as N-methyl N'-nitro-N-nitrosoguanidine and sodium azide. Asafoetida and turmetic extract were found to inhibit microsomal activation dependent mutagenicity of 2-acetamidofluorene. Similar results were also obtained using curcumin and eugenol which are phenolics present in turmeric and clove respectively. These results indicated that some of the spices may ameliorate the effect of environmental mutagens especially present in the food.

Light & electron microscopy of proliferating oval cells during early chemical hepatocarcinogenesis.

Early hepatic changes were studied in male albino rats (70) of Sprague Dawley strain fed a choline devoid diet containing 0.05 per cent w/w AAF (2-acetylaminofluorene) for 12 days. Proliferating periductal and ductal cells appeared in the portal area on days 1 and 3 respectively in the experimental group. On day 7, these cells infiltrated within the sinusoids of adjacent lobules up to the first one or two layers of hepatocytes. Subsequently, these cells extended up to the midzonal region on day 21 and by day 24 the entire lobule was infiltrated. Formation of duct like structures by the proliferating cells was seen on day 21. Ultrastructurally both periductal and ductal cells showed only a few organelles. Periductal and ductal cells are the earliest cells to appear in the portal area in chemically induced hepatocarcinogenesis. Its undifferentiated ultrastructure, may suggest the stem cell nature of these cells.

Factors Influencing the Pathogenicity of Entamoeba Histolytica

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.

Characterization of Tumor Specific Antigens on the Plasma Membrane Surface of Rat Hepatomas lnduced by 3'-Me DAB and ldentification of the Common Tumor Specific Antigens from Rat Hepatomas lnduced by Different Chemical Hepatocarcinogens

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.

A Study for The Effects of UV exposure and UV Exposure After Being Applied by Sunscreens on Hepatic Microsomal Cytochrome Enzyme System and Vitamin D in Newborn Rats / 대한피부과학회지

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).

Effect of vitamin C on the mutagenicity of the carcinogens n-hydroxyphenacetin and n-hydroxy-2-acetylaminofluorene in salmonella typhimurium

The salmonella microsome mutagenesis assay was used to determine the effect of Vit. C on the mutagenic activation of the carcinogens N-hydroxyphenacetin, N-OH-AAF and some related analogues. N-Hydroxyphenacetin was not a directly acting mutagen, but did exert this property in the presence of rodents' liver microsomal fractions. Vit. C inhibited the mutagenic activation of N-hydroxyphenacetin in TA 100, but caused a slight increase in the mutagenic activation of 2-nitrosofluorene, N-hydroxy-2-aminofluorene and N-hydroxy-2-acetylaminofluorene in TA 98. Paraxon, nearly blocked both the mutagenicity of AAF and N-OH-AAF in TA98, mediated by rat liver microsomal fraction