Changes in peroxisomes in preneoplastic liver of rats induced by 3'-methyl-4-dimethylaminoazobenzene.
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Histochemical investigations used the 3,3'-diaminobenzidine reaction to demonstrate the catalase activity and thus variations in numbers of peroxisomes, and electron microscopic examinations were made of hyperplastic liver lesions in rats fed 0.06%3'-methyl-4-dimethylaminoazobenzene. At the 10th week of carcinogen feeding, hyperplastic lesions (hyperplastic foci, areas, and nodules) appeared and advanced to further stages. Most of the foci and some of the areas and nodules showed very low catalase activity and, correspondingly, a small number of peroxisomes. When rats were administered ethyl-alpha-p-chlorophenoxyisobutyrate, most of the foci, areas, and nodules showed a moderate increase in catalase activity and in peroxisome number. Hyperplastic foci seemed to grow larger with time to form hyperplastic areas and/or nodules, mostly accompanying maturation as well as proliferation of the hepatocytes involved. Maturation is well characterized by an increase in the endogenous level of catalase and in the number of peroxisomes, as well as by an enhancement of the responsiveness to ethyl-alpha-p-chlorophenoxyisobutyrate. However, there was a small proportion of lesions in which all cells or some cells did not mature and thus were considered persistently altered. It is suggested that these altered cells serve mainly as intimate precursors of hepatomas. (+info)
Cholangiocarcinomas induced by feeding 3'-methyl-4-dimethylaminoazobenzene to rats. Histopathology and ultrastructure.
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Thirty-three male Sprague-Dawley rats were fed a carcinogenic (0.064% 3'-methyl-4-dimethylaminoazobenzene, 3'-Me-DAB) ground meal normal diet. After 12 weeks the ground meal diet was replaced with a normal pellet diet, and the 30 surviving animals were divided into three equal groups. One group was sacrificed at the twelfth week and the other groups 4 and 8 weeks later. Control animals were also run. Based on previous studies which used "tumor-promoting" diets and 3'-Me-DAB, we expected a less than 100% incidence of predominantly hepatocellular carcinomas. However, we found mucin-producing cholangiocarcinomas in all 30 animals and, in addition, a small hepatocellular component in 3 of the animals. By electron microscopy the intestinal mucosal features of microvillous border cells, goblet cells, and endocrine-like cells were found. We suggest that the tumors produced as described here provide a good animal model of mucin-producing cholangiocarcinomas. (+info)
Marked increases of two kinds of two-exon-skipped albumin mRNAs with aging and their further increase by treatment with 3'-methyl-4-dimethylaminoazobenzene in Nagase analbuminemic rats.
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Nagase analbuminemic rats (NARs) have a 7-base-pair deletion at the 5' splice site of the HI intron of the albumin gene. The level of immunohistochemically albumin-positive hepatocytes is about 1 per 10(5) cells in neonatal NARs, increases with age, and further increases with chronic oral treatment with 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB). The mechanisms involved in the increase in albumin-positive hepatocytes during aging of NARs and their treatment with 3'-MeDAB were analyzed. NARs were found to have four species of albumin mRNA: intact mRNA and those lacking the regions corresponding to exon H, exon G-H, and exon H-I. In 4-week-old NARs, the level of intact albumin mRNA was about 1/4000 of that in normal rats and mRNA lacking the exon H sequence was the major species. In aged and 3'-MeDAB-treated aged NARs, all four species of mRNA increased and the relative proportion of mRNAs lacking two exon sequences to mRNAs lacking one exon sequence was greatly increased, suggesting that aging is associated with changes of the splicing pattern and that 3'-MeDAB treatment enhanced these changes. In aged NARs and 3'-MeDAB-treated aged NARs, there was an increase in the amount of aberrant 60-kDa albumin. The 60-kDa protein could be a translation product of mRNAs lacking two exons, the amount of which increases in aged NARs and 3'-MeDAB-treated NARs. (+info)
A study on the regulation of translocation of glucose transporters during hepatocarcinogenesis induced by 3'-Me DAB.
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The mechanism of glucose transported (GT) expression on the plasma membranes of hepatoma cells in rats induced by 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) was studied. Cytochalasin B binding to plasma membrane fractions from control and 3'-MeDAB group in the absence of cold cytochalasin B showed 9,825 +/- 925 and 30,165 +/- 625 dpm/mg membrane protein. Scatchard plot analysis showed that the GTs present on the plasma membrane fractions in control and 3'-Me DAB groups were 5.0 and 16.0 pmol/mg membrane protein and their Kd values were 151 and 157 nM, respectively. These results suggest that the numbers of GTs in plasma membrane were increased in the 3'-Me DAB group compared to the control group. In contrast, the amounts of GTs in low density microsomal (LDM) fractions measured by a photoaffinity labeling technique using [3H]-cytochalasin B were 31,207 and 11,702 dpm/mg protein in the control and 3'-Me DAB group, respectively. These results suggest that GTs were translocated from LDM to plasma membranes during carcinogenesis. To confirm these results by an independent method 10% SDS-polyacrylamide gel electrophoresis was carried out. Gel slice No. 13 corresponding to MW of 45 kDa from plasma membrane fractions showed increased radioactivities in the 3'-Me DAB group compared to the control group. However, LDM fractions of the 3'-Me DAB group showed decreased radioactivities compared to the control group. Western blot analysis using anti-human RBC GT antibody present in the plasma membranes and LDM fractions from control and 3'-Me DAB groups did not show any significant difference, indicating low cross-reactivity between them. These results indicate that increased glucose transport seems to be more likely due to reciprocal redistribution of GTs between plasma membrane and LDM fractions. (+info)
Appearance of intestinal type of tumor cells in hepatoma tissue induced by 3'-methyl-4-dimethylaminoazobenzene.
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Carcinoma tissues induced by 3'-methyl-4-dimethylaminoazobenzene were investigated both morphologically and biochemically. The most prominent histological pattern was an undifferentiated carcinomatous one. While this type of carcinoma, histologically, appeared to be due to a uniform population of cells, electron microscopic examination revealed that the carcinoma tissue was composed of many types of cells including cells that contained either the brush border or the mucous droplets seen in goblet cells. In addition, tumor cells that contain serotonin-like granules were noticed. An electrophoretogram of alkaline phosphatase in the tissue extract of this type of carcinoma revealed distinctly the presence of its intestinal isozyme. These findings evidently show that carcinoma induced by 3'-methyl-4-dimethylaminoazobenzene includes in addition to the cells differentiated toward hepatocytes or cholangiolar cells, those differentiated toward intestinal epithelial cells. (+info)
Modifications of the expression of liver-specific and non-specific messenger RNAs during azo-dye hepatocarcinogenesis.
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The expression of specific and non-specific rat liver messenger RNAs has been studied during 3'-methyl-4-(dimethylamino)azobenzene (3'-MeDAB) carcinogenesis, using cDNA probes complementary to mRNAs encoding aldolase A and B, L-type pyruvate kinase, albumin, alpha-fetoprotein, transferrin and an unidentified 2.7 X 10(3)-base mRNA. mRNAs specific for undifferentiated cells, such as those encoding aldolase A and the unidentified 2.7 X 10(3)-base species were re-expressed very early, being easily detectable at the 1st week of 3'-MeDAB treatment. They reached a maximum of expression at the 4th week. Simultaneously the levels of aldolase B and L-type pyruvate kinase mRNAs dramatically decreased as compared to controls, but remained responsive to induction by a high-carbohydrate diet. Albumin and transferrin mRNA levels were only slightly modified in the course of the carcinogenic diet. At the terminal stage of hepatocarcinogenesis, i.e. in malignant hepatoma cells, expression and inducibility of aldolase B and L-type pyruvate kinase mRNAs were similar to those in normal adult rats while mRNAs specific for undifferentiated or foetal stages were also synthesized. The very early changes in gene expression for aldolases A and B, L-type pyruvate kinase and the 2.7 X 10(3)-base mRNA species could indicate that carcinogenic diet modifies gene control mechanisms long before inducing hepatoma. (+info)
Promotion of growth and differentiation of rat ductular oval cells in primary culture.
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Oval cells emerging in rat liver at the early period of 3-methyl-4-dimethylaminoazobenzene treatment constitute a mixed epithelial cell compartment with respect to alpha-fetoprotein (AFP) and cytokeratin differential expression, and include a subpopulation which exhibits a phenotype intermediate between ductular cells and hepatocytes (Germain et al., Cancer Res., 45:673-681, 1985). In the present study we have examined the developmental potential of ductular oval cells in primary culture and after in vivo transfer. The use of monoclonal and polyclonal antibodies directed against cytokeratins of Mr 39,000 (CK39), 52,000 (CK52), and 55,000 (CK55) and vimentin, and also monoclonal antibodies against exposed surface components of oval cells (BDS7) and normal hepatocytes (HES6) allowed us to establish the ductular phenotype of the oval cells. A highly enriched preparation of oval cells was obtained by perfusion/digestion of the liver with collagenase, treatment of the cell suspension with trypsin and DNase, selective removal of hepatocytes by panning using the anti-HES6 antibody, and cell separation by isopyknic centrifugation in a Percoll gradient. The procedure yielded about 8 x 10(7) cells, of which 95% expressed CK39, CK52, and BDS7, 84% gamma-glutamyl transpeptidase, and 5% albumin and AFP. The primary response of cultured oval cells to various combinations of growth and differentiation promoting factors was evaluated with respect to their capacity to initiate DNA synthesis as measured by [3H]thymidine labeling from day 1 to 3, and/or to produce albumin and AFP and express tyrosine aminotransferase. Culture in the presence of either serum or clot blood extract resulted in a low proliferative activity with less than 5% of the nuclei being labeled. Over a 5-day period, fusion of a large portion of the oval cells led to multinucleated cells. When the cells were cultured in the presence of an elaborate combination of supplements [minimum essential medium containing 1 mM pyruvate, 0.2 mM aspartate, 0.2 mM serine, 1 mM tyrosine, 1 mM proline, 1 mM phenylalanine and supplemented with 20% clot blood extract, 10 ng/ml oxidized bile acids, 17 microM bilirubin, 10 ng/ml cholera toxin, 1 microM dexamethasone, 2.5 micrograms/ml insulin, 50 mM beta-mercaptoethanol, and 5 micrograms/ml transferrin (medium MX)], the labeling index increased to around 30% and the level of cell fusion greatly decreased. The addition of dimethyl sulfoxide further enhanced the initiation of DNA synthesis, while sodium butyrate acted as an inhibitor.(ABSTRACT TRUNCATED AT 400 WORDS) (+info)
Cellular expression of alpha-fetoprotein gene and its relation to albumin gene expression during rat azo-dye hepatocarcinogenesis.
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During hepatocarcinogenesis, alpha-fetoprotein (AFP) synthesis may be dramatically increased while albumin synthesis is frequently decreased. Therefore, a reciprocal modulation between both gene expressions has been hypothesized. In this work, we combined in situ hybridization and immunoperoxidase on parallel liver tissue sections in order to analyze at the cellular level, AFP gene expression and its relation to ALB gene expression in both early and neoplastic lesions induced by 3MeDAB in the rat. In early lesions, cell populations were heterogenous as regards AFP expression. High levels of AFP transcripts were detected both in oval type cells and in a subset of basophilic hepatocytes within preneoplastic lesions. In these two highly AFP-expressing cell populations, significant levels of ALB transcripts were concomitantly detected. In the majority of altered hepatocytes, no AFP expression was detected while the level of ALB expression was decreased. In neoplastic lesions, AFP expression was strikingly heterogenous and independent from the degree of morphological differentiation. No evidence of reciprocal modulation with ALB gene expression could be assessed. In both preneoplastic and neoplastic lesions, a few altered hepatocytes displayed significant levels of AFP transcripts while no corresponding protein could be detected; such a discrepancy was not observed for ALB. This work shows that during 3MeDAB hepatocarcinogenesis, AFP gene activation occurs in heterogenous cell populations and according to different cellular patterns. Our observations lend no support to the hypothesis of a reciprocal modulation between AFP and ALB gene expressions during rat azo-dye hepatocarcinogenesis. (+info)