Effect of antioxidant vitamins A, C, E and their analogues on azo-dye binding protein in liver of rats treated with p-dimethylaminoazobenzene.

p-Dimethylaminoazobenzene (DAB) is an azo-dye and known to cause liver tumour in rats. Azo-dye binding protein is a specific cytosolic protein involved in the translocation of azo-dye carcinogen metabolites from liver cytoplasm into the nucleus. Administration of vitamin A (40,000 and 50,000 IU), L-ascorbic acid (500 and 1,000 mg) and vitamin E succinate (200–500 mg) reduced the amount of azo-dye binding protein in liver of rats treated with DAB. Supplementation of high doses of vitamin A acetate, vitamin A palmitate, sodium ascorbate, ascorbyl palmitate and vitamin E acetate had no effect on the quantity of azo-dye binding protein in liver. When the vitamin mixture was given, the level of azo-dye binding protein decreased in the liver at all the studied doses, which may be due to their synergistic effect.

Protein methylation in cellular proliferation and differentiation: Non-histone nuclear methyl acceptor protein(s) during 3'-methyl-4-dimethylaminoazobenzeneinduced hepatocarcinogenesis

An accelerating effect of methyl-deficient diet (MDD) on hepatocarcinogenesis and methylation pattern of nuclear protein(s) by S-adenosylmethionine: protein arginine N-methyltransferase (protein methylase I, PM-I) have been studied with 3'-methyl-4-dimethyl- aminoazobenzene(MeDAB)-treated rats. The MDD+MeDAB-fed group produced typical cancer cells in the liver almost two weeks earlier than the control synthetic diet (CSD)+MeDAB-fed group. Protein methylase I (PM-I) activity in the livers of MDD alone fed rats began to increase at around 2 weeks after MDD-feeding, reaching a peak at 4 weeks and declining thereafter. When nuclei isolated either from normal livers or from cholangiocarcinoma cells were incubated with PM-I preparation from normal liver, 16 and 23-kDa nuclear proteins were the major methylated proteins, regardless of the source of the nuclei. However, when the above mentioned nuclei were incubated with PM-I preparations either from MDD alone fed livers or MDD+ MeDAB-induced cholangiocarcinoma cells, the methylation of 23-kDa protein was not detected. The result suggests that there is a hitherto-unknown PM-I specific to 23 kDa nuclear protein which was lost during methyl deficient diet feeding and hepatocarcinogenesis. The N-terminal 20 amino acids sequence of the 23-kDa protein was found to be (1)Gly-Val-Pro-Leu-(5)X-Arg-Leu-Phe-Asp-(10)His-Ala-Met-Leu-Gln-(15)Ala -His-Arg-Ala-His-(20)Glu, having 94.7% sequence homology with human chorionic somatomammotropin precursor A and B.

Expressions of c-fos and c-myc genes during 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB)-induced rat hepatocarcinoma

We investigated the expression of the growth-related nuclear proto-oncogenes, c-fos and c-myc, in early preneoplastic regions and tumor nodules of 3'-MeDAB induced rat hepatocarcinoma. To amplify the levels of these transcripts, we gave cycloheximide (100 mg/kg B.W. i.p.) to each group of rats. The elevated levels of the 2.2 kb c-fos and 2.4 kb c-myc transcripts appeared as early as the 2nd week after feeding on the 3'-MeDAB diet and lasted through the 4th; 6th weeks and tumor. Southern blot analysis indicated that gross amplification or rearrangements were not observed in DNA of the preneoplastic livers and hepatoma nodules. We also measured the rate of the incorporation of [3H] thymidine into hepatic DNA in order to monitor the rate of cell proliferation occurring at the early preneoplastic periods. We have found that the rate of [3H] thymidine incorporation corresponds to the elevated levels of c-fos and c-myc transcripts in the precancerous stages. This finding suggests that the elevated expressions of c-fos and c-myc may result from the continuous cell proliferative stimuli generated in the carcinogen altered cells, which is essential to the initiation and promotion of chemical hepatocarcinogenesis.

The Effect of Copper on 3'-Methyl-4-dimethylaminoazobenzene Induced hepatic Carcinogenesis

To elucidate the effect of copper on the 3'-methyl-4-dimethylaminoazobenzene(3'-MeDAB) induced hepatic carcinogenesis, Sprague-Dawley rats were divided into 4 groups according to 3'-MeDAB and copper administration: I. noraml control, II. copper only, III. 3'-MeDAB only, IV. 3'-MeDAB plus copper. The animals of groups III and IV were fed experimental diet containing 0.06% 3'-MeDAB. Copper was administrated intraperitoneally in a dose of 0.5 mg, twice a weak. Animals were sacrificed at different intervals. Liver weight, hepatic copper content and gross and microscopical changes of the liver were examined and the cell kinetics of various lesions in the hepatic carcinogenesis was studied by applying the immunohistochemical method for bromodeoxyuridine(BrdU). The hepatic copper content was significantly increased in animals given copper but returned to the normal value after cessation of adminstration. 3'-MeDAB administration caused oval cell proliferation and produced hyperplastic nodules, cholangiofibrosis and carcinoma of the liver. Simultaneous administration of copper did not alter the incidence of 3'-MeDAB induced lesions, except for carcinoma. The liver weight and the size of hepatic nodules and masses were smaller in group IV than in group III. The liver weight as well as the nodularity and the mass formation continued to increase affect cessation of 3'-MeDAB administration. Copper did not affect the BrdU labelling indices of the hepatic lesions induced by 3'-MeDAB. The oval cell proliferation and the BrdU labelling indices of the oval cell and the hyperplastic nodule were decreased, but the incidence of cholangiofibrosis and its BrdU labelling index were still elevated after cessation of 3'MeDAB administration. These findings indicate that copper could delay the developement of 3'-MeDAB induced hepatic lesions, but not suppress, since copper does not stay long enough to accumulate in the rat liver, and that copper could not affect the proliferation of 3'-MeDAB induced hepatic lesions once developed.

The Effect of Ethanol on 3'-Methyl-4-dimethylaminoazobenzene Induced Carcinogenesis in Rat Liver

This study is aimed to elucidate the biological nature of the precancerous lesions and to evaluate whether the ethanol alters 3'-Methyl-4-dimethylaminoazobenzene (3'-Me-DAB) induced experimental hepatocarcinogenesis. A total of 108 Sprague-Dawley male rats were used for the experiment and divided into 6 groups according to 3'-Me-DAB and ethanol administration. Administration of the drugs were carried out daily by nasogastric tube insertion and the animals were sacrificed at different interval. A part of right lateral lobe was prepared for the histological examination. Cell kinetics of the immunohistochemical method for bromodeoxyuridine (BrdU). The administration of 3'-Me-DAB induced oval cell proliferation, hyperplastic nodule, cholangiofibrosis and carcinoma in the liver. The mean labelling indices, the percentages of BrdU labelled cells, of hepatocytes were increased by administration of 3'-Me-DAB, only to reverse after cessation of the drug (2.58 vs 0.61). The labelling indices of the oval cells were also affected by the administration and cessation of 3'-Me-DAB (11.41 vs 4.48). In contrast, the cholangiofibrosis did not decrease but were still increasing following cessation of 3'-Me-DAB administration (4.37 vs 5.17 and 8.25 vs 11.29). These finding that the hyperplastic nodule and particularly the cholangiofibrosis have an autonomous proliferative potential and are definite precancerous lesions in the experimental hepatocarcinogenesis. Short term administration of ethanol decreased the incidence of development of the precancerous lesions, but did not affect the labelling indices in all the pathologic lesions of hepatocarcinogenesis.