Excretion of alpha-foetoprotein in the urine of rats during exposure to 3'-methyl-4-dimethylaminoazobenzene.
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Female rats were maintained on standard laboratory diet, Miller's diet or Miller's diet containing 3'MeDAB. Animals fed standard or Miller's diet did not excrete alpha-foetoprotein (AFP) in their urine. Early appearance of AFP was demonstrated by examining the urine of rats on the 3'MeDAB regimen. The incidence of positive urine samples was high between the 5th and 7th week of the experiment. It thereafter declined, but from the 3rd month it steadily rose and reached a maximum of 80% at about 10 months. Though urinary excretion of AFP was irregular in individual animals, several positive urine samples were obtained from all rats followed for more than a few months. The urine of 90% of hepatoma-bearing rats contained AFP at the time of killing. The incidence of elevated serum AFP levels as determined by immunodiffusion, increased with the duration of the experiment, but was still only 70 percent in rats fed 3'MeDAB for over 34 weeks. The severity of the hepatic alterations, as well as hepatocytic uptake of [3H]thymidine, increased with time. The serum of animals fed the standard diet was negative, whereas AFP was very infrequently detected in the serum of rats given Miller's hypoprotein diet. The results demonstrate that, in a population exposed to hepatocarcinogenic agent, the recurring detection of urinary excretion of AFP is a useful indicator of the high risk of developing hepatomas. (+info)
Functional changes in rat liver mitochondria on administration of 2-methyl-4-dimethylaminoazobenzene.
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Administration of 2-methyl-4-dimethylaminobenzene in the diet (0.1%, w/w) for 85-90 days doubled the content of mitochondria in the livers of rats. The azodye was covalently bound to liver proteins, and about 15% of the amount found in liver was associated with the mitochondrial fraction. Mitochondria isolated from the livers of azodye-fed animals showed drastically lowered ability to oxidize NAD+-linked substrates. The inhibited electron-transfer step was the reduction of ubiquinone. The organelles showed a large increase in succinate oxidase activity. The activity of cytochrome oxidase and the content of cytochrome aa3 were substantially higher in these organelles. Azodye-fed animals showed depressed serum cholesterol concentrations. The content of ubiquinone in liver also registered a small increase. (+info)
Nuclear matrix antigens in azo dye-induced primary rat hepatomas.
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Polyvalent antisera, monoclonal antibodies, and immunotransfer methodology have been used to identify and characterize a group of chromosomal protein antigens which appear during azo dye hepatocarcinogenesis. Experiments were designed to probe for the location and placement of antigens in chromatin according to solubility and possible DNA-binding properties. The majority of nuclear antigens were associated with high-speed DNA-containing pellets after ultracentrifugation of chromatin solubilized with denaturing buffers containing 6 M guanidine-HCl:2% sodium dodecyl sulfate, or 2 M NaCl:5 M urea. The addition of 2-mercaptoethanol or dithiothreitol to guanidine-HCl or sodium dodecyl sulfate solutions resulted in solubilization of nearly all antigens from the DNA pellets, suggesting the presence of complexes (protein:protein and/or DNA:protein) cross-linked with sulfhydryl linkages. Preparation of nuclear matrix from the primary hepatomas under several kinds of conditions indicated these antigens to be components of the residual nuclear matrix, envelope, and/or associated structures. Two-dimensional gel analysis showed most antigens to exist in a range of isoelectric forms, suggesting posttranslational modifications. Studies with monoclonal antibodies prepared to these proteins revealed extensive antigenic homology among the members comprising these fractions. Our results document antigenic differences in the nuclear matrix proteins of primary tumors and their normal tissue counterparts. (+info)
Unscheduled DNA synthesis induced by procarcinogens in suspensions and primary cultures of hepatocytes on collagen membranes.
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Unscheduled DNA synthesis was induced by procarcinogens in freshly isolated suspensions and primary cultures (6 days old) of hepatocytes on collagen membranes. Incorporation of [3H]thymidine in the presence of hydroxyurea was used to measure unscheduled DNA synthesis. When hepatocellular DNA was isolated on cesium chloride gradients, significant levels of unscheduled DNA synthesis were measured. Similar concentrations of procarcinogens elicited higher levels of unscheduled DNA synthesis in hepatocellular suspensions than in primary cultures. The results demonstrate that hepatocytes cultured on collagen membranes can metabolize chemical carcinogens. Suspensions of freshly isolated hepatocytes, however, are more active in procarcinogen metabolism than are those of primary cultures. The selective advantages of the two systems of hepatocytes can be utilized for the establishment of short-term in vitro screening systems of mutagens and carcinogens. (+info)
Binding of 3'-methyl-N,N-dimethyl-4-aminoazobenzene metabolites to rat liver cytosol proteins and ligandin subunits.
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Twenty min after i.p. administration of 3'-[14C]methyl-N,N-dimethyl-4-aminoazobenzene in corn oil to rats, 0.73% of administered radioactivity was present in the liver. Only 0.45% of radioactivity present in liver was recovered in the nuclear fraction, whereas 25% was present in the cytosol fraction. Twenty-seven % of cytosolic radioactivity was trichloroacetic acid precipitable, and 2% was immunoprecipitable with monospecific anti-rat liver ligandin immunoglobulin G. After 3 hr of administration, 3.2% of administered radioactivity was present in the liver, 40% of which was in the cytosol. Although 59% of radioactivity present in liver cytosol was trichloroacetic acid precipitable as compared to 27% at 20 min, the radioactivity precipitated by anti-ligandin immunoglobulin G was still 2%. When liver cytosol obtained from rats after 20 min of 3'-[14C]methyl-N,N-dimethyl-4-aminoazobenzene administration was fractionated on a Sephadex G-75 column, three peaks of radioactivity were observed. When cytosol was subjected to sodium dodecyl sulfate gel electrophoresis and fluorography, radioactivity was mainly associated with 5 proteins with molecular weights of 88,000, 47,000, 41,000, 31,000, and 22,000. When the immunoprecipitate obtained from cytosol with anti-ligandin immunoglobulin G was subjected to sodium dodecyl sulfate gel electrophoresis and fluorography, radioactivity was exclusively associated with the subunit of ligandin with a molecular weight of 22,000. Approximately 90% of the radioactivity in the immunoprecipitate was covalently associated with this subunit. These studies reveal that 3'-methyl-N,N-dimethyl-4-aminoazobenzene or its metabolites are selectively bound to the subunit of ligandin with a molecular weight of 22,000 and four other cytosol proteins in vivo. (+info)
Inhibition of monoamine oxidase by 3'-methyl-4-dimethylamino azobenzene (3'-me-DAB) in rat liver mitochondria.
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Effects of 3'-Me-DAB on MAO in rat liver mitochondria, in vitro, were investigated. 3'-Me-DAB at a concentration of 1 x 10(-5) M inhibits MAO activity about 40%, and this inhibition recovered to the control value after dialysis overnight against 0.001 M phosphate buffer. MAO activity was inhibited in an apparently competitive fashion by 3'-Me-DAB. These results indicate that 3'-Me-DAB binds to mitochondrial MAO with a weak affinity in vitro. The Km value toward benzylamine was 220 microM using both the mitochondria from the liver of rats fed a basal diet and those from rats ingesting 3'-Me-DAB. The activity of these enzyme preparations did not revert after dialysis to the control values of rats fed a basal diet. The titration experiment of MAO by pargyline suggests that the decrease of MAO activity, in vitro, is mainly due to the decrease of active MAO molecules in these mitochondrial preparations from livers of rats ingesting 3'-Me-DAB. (+info)
Biphasic change of proton magnetic relaxation times during azo-dye hepatocarcinogenesis.
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For the first time, change in the proton longitudinal relaxation times (T1) of rat tissues has been examined throughout the whole process of azo-dye hepatocarcinogenesis. Two maxima of the T1 values were observed for liver, on Day 60 and after Day 120, and these changes correlated well with the changes in water content. The first peak was ascribed to the immature hepatocytes of hyperplastic nodules, and the second peak to the developed hepatoma cells. The significance of the change in T1 values as a preneoplastic change is discussed. (+info)
Histochemical and cytochemical study of butyrylcholinesterase activity in rat hepatocellular carcinomas induced by 3'-methyl-4-dimethylaminoazobenzene.
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The activity of butyrylcholinesterase (BCHE), a liver fetal isozyme (Zone L-V) of a nonspecific esterase, was studied histochemically and cytochemically in rat hepatocellular carcinomas induced by 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB). In normal adult rats, BCHE activity was very prominent in cells of the intestinal mucosa but was not detectable in the liver. On the other hand, in fetal rat liver, a few cells scattered throughout the organ were BCHE positive. 3'-Me-DAB induced poorly differentiated hepatocellular carcinomas showing an intense BCHE activity, especially in areas consisting of small tumoral cells proliferating in a sheet-like pattern. Surrounding noncancerous liver tissue was completely devoid of reaction products. Less-differentiated trabecular hepatocellular carcinomas also showed a positive reaction. On the other hand, well-differentiated hepatocellular carcinoma and hepatocellular carcinoma with an adenomatous pattern were barely stained, while areas of cholangiofibrosis were usually negative. Thus, in confirmation of a previous report, BCHE appears to be a positive marker of poorly differentiated hepatocellular carcinomas induced by 3'-Me-DAB. By electron microscopy, reaction products were demonstrated in the cisternae of the endoplasmic reticulum, in the nuclear envelopes, and sometimes on the cell surface of undifferentiated tumoral cells. The significance of the appearance of BCHE activity in hepatocellular carcinomas induced by 3'-Me-DAB is discussed. (+info)