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The changes in glycosaminoglycans in livers of rats with 3'-methyl-4-dimethylaminoazobenzene-induced hepatic cancer were examined and compared with those in fetal liver. The incorporation of 35S into sulfated glycosaminoglycans in hepatic cancer tissue was also studied after i.p. injection of Na(2)35SO4. The major component of glycosaminoglycans in healthy adult rat liver was heparan sulfate (61.7%), with hyaluronic acid (21.1%), dermatan sulfate (13.1%), and chondroitin sulfate (4.0%) as minor components. The quantities of all of the examined glycosaminoglycans were higher in tumors than in normal liver, but chondroitin sulfate and hyaluronic acid were more prevalent in the tumors (about 51 and 7 times higher, respectively). As a result, the share of heparan sulfate was decreased (37.1%) in the tumors. The high content of chondroitin sulfate (about 30 times) and the decreased share of heparan sulfate (29.0%) were also observed in fetal liver. The incorporation of 35S into individual glycosaminoglycans varied markedly. Approximately 90% of the label in glycosaminoglycans of healthy liver was found in a heparan sulfate fraction 4 hr after injection. In hepatic cancer tissue, however, 35S incorporation into both chondroitin sulfate and dermatan sulfate fractions was increased about 6.5- and 5.6-fold, respectively, of those in healthy liver.

Unscheduled DNA synthesis was observed in primary rat liver cell cultures treated with members of five different classes of chemical procarcinogens requiring enzymatic activation as well as with a direct-acting carcinogen. In total, ten carcinogens and one related analog not commonly accepted as carcinogenic were active, while one weak carcinogen and four noncarcinogens were inactive. The production of unscheduled DNA synthesis by this spectrum of chemical carcinogens indicates that these cultures have substantially retained the metabolic capability of liver for activating diverse procarcinogens. Thus, such cultures may be useful for detecting the ability of chemicals to interact with DNA and, thereby, assigning them priority for consideration as potential cancer-causing agents.

Male rats were given single doses p.o. of 3'-methyl-4-dimethylaminoazobenzne (3'-MeDAB), either alone or at the same time as single s.c. injections of cycloheximide. They were killed either 24 or 48 hr after treatment or at intervals up to 21 months, and their hepatic tissues were examined by electron density, combined treatment with both chemicals failed to produce this acute toxic effect. However, the fine structure of the hepatic cells of rats given this combined treatment with cycloheximide and 3'-MeDAB closely resembled that obtained by chronic exposure to carcinogenic azo dyes. Of the changes thus produced, the granular endoplasmic reticulum in particular became permanently altered, both quantitatively and morphologically. Other persistent changes included mitochondrial abnormalities and glycogen depletion. Cycloheximide appears to protect the liver cell against the nonspecific acute toxic action of 3'-MeDAB, while facilitating the expression of effects that may possibly be associated with the carcinogenic action of this azo dye. Although this experimental model does not result in the appearance of tumors, it demonstrates that a single exposure to a carcinogen may induce permanent changes that are similar to those observed during carcinogenesis.

The effect of portacaval shunt on hepatocarcinogenesis was studied in rats fed 3'-methyl-4-dimethylaminoazobenzene. Portacaval anastomosis resulted in a decrease of hepatocarcinogenesis as reflected by a delay in the early peak of alpha-fetoproteins, an absence of late appearance of alpha-fetoproteins, and a significantly lower incidence of tumors than in nonshunted rats. Reduction of hepatocarcinogenesis in shunted rats was associated with a decrease of the binding of 3'-methy-4-dimethylamioazobenzene metabolites to liver proteins. This effect seemed to be related to modifications of carcinogen-metabolic pathways. While the detoxifying azoreductase activity was not affected by portal diversion, the activating pathway leading to the binding of 4-dimethylaminoazobenzene metabolites to DNA, a major step for cell carcinogenesis that is mediated by microsomal enzymes, was decreased in shunted rats to about 50 percent of control values. The decrease of liver weight that occurred in shunted rats without loss of body weight produced a very significant reduction of the total capacity of liver to activate 4-dimethylaminoazobenzene while the total capacity of detoxification remained unchanged. This could be a direct consequence of portacaval anastomosis, as has been shown for other microsomal enzymes.

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.

Enzyme deviation patterns were examined in primary rat hepatomas induced by short-term sequential administration of two chemical carcinogens from among 2-fluorenylacetamide (FAA), diethylnitrosamine (DENA), and 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) or by FAA or 3'-Me-DAB followed by phenobarbital as a promoter. The purpose was to discern how the patterns are influenced by different administration schedules of carcinogens and which of the two carcinogens in the sequence affects the pattern more. Biochemical differentiation of hyperplastic hepatic nodules and hepatomas was determined by simultaneous assays of activities and isozyme composition of glucose-adenosine triphosphate phosphotransferase, pyruvate kinase, glucose-6-phosphatase, fructose-1,6-bisphosphatase, and gamma-glutamyltransferase with consideration of histological classification of nodules and tumors. Poorly differentiated hepatomas were predominantly induced by 3'-Me-DAB followed by FAA or DENA except for hepatomas induced by 3'-Me-DAB followed by phenobarbital, which were mainly well and moderately differentiated; well and moderately differentiated hepatomas were predominantly induced by FAA followed by 3'-Me-DAB or phenobarbital. The degree of enzyme deviation of the hepatomas induced by DENA as the first carcinogen was intermediate between those of hepatomas induced by FAA or 3'-Me-DAB, although the degree tended to increase with increased dose or term of DENA. These results indicate that deviations of some enzymes, such as pyruvate kinase and fructose-1,6-bisphosphatase, as well as histological differentiation of the primary hepatomas are more strongly influenced by the first carcinogen than by the second under our administration schedules and that the degree of enzyme deviation shown by hepatomas produced by a particular carcinogen treatment regimen principally related to the potential of that regimen to induce the more anaplastic tumors.

The effect of hepatocarcinogen administration in vivo on the induction of pleiotropic drug resistance was studied in primary monolayer cultures of adult rat hepatocytes using a cytotoxicity assay in vitro. Dietary 2-acetylaminofluorene, 3'-methyl-4-dimethylaminoazobenzene, aflatoxin B1, ethionine, and diethylnitrosamine rapidly induced resistance to doses of Adriamycin, methotrexate, cycloheximide, and aflatoxin B1 which were cytocidal to normal hepatocytes from untreated rats. Up to 95% of some hepatocyte preparations became drug resistant before any new hepatocyte phenotypes could proliferate. Drug resistance was measured at 24 h after initiation of 2-acetylaminofluorene feeding and remained stable throughout the 16 wk of carcinogen exposure. When limited carcinogen exposure was followed by a return to a basal non-carcinogen-containing diet for many months, the hepatocytes in the resultant hepatocellular carcinomas also displayed pleiotropic drug resistance, and the cells of the peritumorous liver did so to a lesser extent. Drug resistance was not induced by chronic administration of the tumor promoters phenobarbital, choline-deficient diet, phorbol, nor with 2,3,7,8-tetrachlorodibenzo-p-dioxin, but was induced to a variable extent by three hepatotoxins (ethanol, methotrexate, carbon tetrachloride). Whereas the early appearing drug resistance appears to be an adaptation of the liver to the presence of a toxic carcinogen, the late resistance which does not disappear after withdrawal of the inducing carcinogen may be a constitutive characteristic of chemically induced hepatocellular carcinomas.

Two radiolabeled hepatocarcinogens, N,N-dimethyl-4-aminoazobenzene (DAB) and 3'-methyl-N,N-dimethyl-4-aminoazobenzene (3'-Me-DAB), were rapidly cleared from the blood of rats after i.v. administration, with half-lives of 40 and 70 sec, respectively. Rates of hepatic uptake and biliary secretion of [14C]-3'-Me-DAB were double that of [14C]DAB within 30 min of administration. Two hr after azo dye injection, the hepatic output into bile of [14C]-3'-Me-DAB-derived radioactivity was three times that of [14C]DAB. Fifty and 75% of the total 3'-Me-DAB-derived radioactivity was recovered in blood, liver, and bile 30 and 120 min after injection while only 30 to 40% of the administered [14C]DAB-derived radioactivity was recovered at these times. We postulate the existence of an extrahepatic azo dye accumulation site which may compete with the ability of the liver to clear azo dye from the circulation and which releases 3'-Me-DAB-derived radioactivity more readily than that of DAB. Azo dye metabolites were isolated from liver, bile, and blood. The chromatographic pattern of liver metabolites generated in vivo by rats which received either hepatocarcinogen was obtained and compared with that of biliary metabolites. With either azo dye, some metabolites were located exclusively in the liver, some were secreted immediately into bile, while others were present in both liver and bile, indicating selectivity in biliary excretion.

We have investigated the appearance of specific nonhistone proteins during azo dye-induced hepatocarcinogenesis in the rat. Groups of animals fed azo dye-containing diet were sacrificed at approximately 3-week intervals, portions of their livers were examined histologically, and the remaining material was fractionated into chromatin and cytoplasmic fractions. Livers of the azo dye-fed animals exhibited histological changes that have been classically attributed to the course and development of cancer; by 28 to 30 weeks of treatment, nearly all animals had developed hepatomas. Heterogeneous rabbit antisera were prepared to dehistonized chromatin from several azo dye-induced hepatomas. These antisera were then used to assess various chromatins for the appearance of antigens specific for neoplasia during inducing carcinogenesis using immunodetection of antigens separated electrophoretically and transferred to nitrocellulose. Changes in the immunoreactivity of liver chromosomal proteins during carcinogen treatment were evident after 3 weeks, and the antigenic profiles of various chromatin samples gradually assumed the characteristics of the hepatoma. The transformation was accompanied by qualitative changes in chromosomal protein antigens, and although these antigenic species were not directly quantitated, noticeable enrichment of tumor-specific species occurred with treatment time. Immunotransfer assays of cytoplasmic fractions indicated most antigens to be specific for chromatin. Normal tissue chromatin exhibited minimal immunoreactivity, and slightly more antigenic homology was noted with regenerating liver and most transplantable tumor chromatins. Interestingly, the transplantable tumor Walker 256 carcinosarcoma was highly enriched in antigens recognized by antisera to azo dye hepatoma dehistonized chromatin. These studies establish a define chronological correlation between the chemical induction of cancer and sequential changes in the immunological specificity of nonhistone protein antigens.

The hepatocarcinogenicity of 2-methyl-N,N-dimethyl-4-aminoazobenzene, previously shown to be noncarcinogenic in adult rats in the absence of further treatment, was observed by following a 1- to 6-week period of feeding this dye to weanling rats with the dietary administration of 0.05% phenobarbital for up to 70 weeks. Many large hepatocellular carcinomas developed in the phenobarbital-treated animals by 72 weeks, whereas a very small number of tiny neoplastic nodules, including one carcinoma, were seen in the rats not given this drug. This study suggests that the use of promoting agents, following the short-term administration of weak carcinogens for the liver, can be useful in demonstrating the initiating activity of such compounds. This system may be useful in the identification of such agents in the environment.

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.

Using an immunofluorescent technique, we demonstrated the production of rat embryonic-type hemoglobin in extra-medullary hematopoietic cells, which appeared in the liver of adult Wistar rats fed a diet containing 0.06% 3'-methyl-4=dimethylaminoazobenzene. Specific anti-rat embryonic hemoglobin serum was prepared by immunizing a rabbit with hemoglobin from a 15- or 16-day-old Wistar rat fetus and absorbing with adult rat liver hemogenate and red blood cells. The appearance of hematopoietic foci was observed at both precancerous and cancerous stages during 3'-methyl-4-dimethylaminoazobenzene-induced liver carcinogenesis. At the cancerous stage, almost all these foci were observed in poorly differentiated hepatocellular carcinomas. The hematopoietic cells seen at both stages were stained positively with antiembryonic hemoglobin serum, but red blood cells and other types of cells in the liver did not show this specific fluorescence. These results together with earlier observations on the appearance of fetal characteristics strongly suggest that a fetal environment prevails in the adult rat liver during hepatic carcinogenesis.

Administration of 2-acetylaminofluorene to rats for 12 weeks induces hyperplastic nodules (HPNs) and later well-differentiated hepatocellular carcinomas (HCCs) in the liver. Total cellular proteins from normal liver, HPN, and HCC were analyzed by two-dimensional gel electrophoresis with a high resolution. Several hundred polypeptides were well resolved as seen by Coomassie blue staining, forming a reproducible and characteristic pattern for each tissue. The polypeptide patterns were very similar among normal liver, HPN, and HCC. Especially the proteins of HPN and HCC were almost indistinguishable. These neoplastic lesions, however, were clearly different from control liver in that a new spot p35-6.6 (designated by molecular weight X 10(-3) and pl) appeared, and five polypeptides, p57-6.9, p57-6.7, p26-6.9, p26-6.6, p26-6.4, increased dramatically in amount as compared with normal liver. These last three spots were found to be a new type of glutathione S-transferase as judged from the specific binding to the antibody. The same changes in polypeptide pattern were found in HCCs induced by other chemical carcinogens, diethylnitrosamine and 3'-methyl-4-dimethylaminoazobenzene, but not in regenerating and neonatal livers. Fetal liver showed a rather different pattern than adult liver, but only p26-6.6 was increased among the spots characteristic of HPN and HCC. Protein phosphorylation was also examined for these cells by incubating tissue slices with 32PO4. After alkali treatment of the gels to eliminate serines phosphorylation, several dozens of phosphoproteins were clearly detected. The patterns of the labeled spots were again very similar among control liver, HPN, and HCC. Only the intensity of a spot designated p57-6.6 increased markedly in both HPN and HCC. This spot was further resolved by an expanded pH gradient into four distinct spots, the major one of which contained phosphothreonine. Similar changes in phosphorylation were noted in hepatomas induced by diethylnitrosamine and 3'-methyl-4-dimethylaminoazobenzene but not in regenerating, fetal, and neonatal livers. These changes are discussed in terms of gene expression relevant to malignant transformation of hepatic cells.

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.

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)

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.

We have developed a new strain of rats (resistant rat) which exhibit resistance against the carcinogenic action of several carcinogenic compounds. In the present study, we compared the ability of resistant rat liver to activate 3'-methyl-4-dimethylaminoazobenzene and 2-acetylaminofluorene to highly reactive metabolites, which can covalently bind to DNA, with that of carcinogen-sensitive parent strain rats (sensitive rat), using in vitro DNA binding assay. The covalent binding of these carcinogens to calf thymus DNA, catalyzed by the hepatic 9000 x g supernatant fraction from resistant rats pretreated with 3-methylcholanthrene, was about one-half of that catalyzed by the 9000 x g supernatant from sensitive rats which had received the same treatment. These results suggest that the ability of resistant rat liver to activate the carcinogens decreases compared to sensitive rat liver. Further experiments revealed that this decreased activation of the carcinogens in resistant rat livers is due to a low inducibility of 3-methylcholanthrene-inducible forms of cytochrome P-450 mRNA. The hepatic cytosolic Ah receptor concentrations in resistant rats were shown to be significantly lower than those of sensitive rats. Scatchard plot analysis demonstrated, however, that there is no significant difference between the affinity of Ah receptors for 2,3,7,8-tetrachlorodibenzo-p-dioxin in these two strains. These data implicate that the hepatic Ah receptor level may be an important factor in determining carcinogen sensitivity in rats.

Plasma membranes (PM's) were isolated from primary hepatomas induced in Wistar rats by 3'-methyl-4-dimethylaminoazobenzene, from nonhepatomal regions of the same rat livers, and also from various normal rat livers, including the resting and regenerating livers of adult rats and developing livers of postnatal rats. Phospholipid analyses of these PM preparations revealed the following differences in the hepatoma PM in comparison with those of the PM of normal adult resting livers: (a) decrease in the content of total phospholipids; (b) large increase in plasmalogen content; (c) relative increase of sphingomyelin and ethanolamine phospholipids, and the decrease of choline phosphoglycerides, i.e., decrease of the ratios of choline phosphoglycerides to choline phosphosphingoside and of choline-containing phospholipids to ethanolamine-containing phospholipids; (d) decrease in phosphatidylserine and phosphatidylinositol. The phospholipid composition of the PM's from normal growing livers showed definite decreases in phosphatidylserine and sphingomyelin and increases in ethanolamine phospholipids; however, no significant alteration was observed in plasmalogen content in comparison to the PM of normal adult resting livers. The PM from the nonhepatomal regions of the hepatoma-bearing livers did not show those differences observed in hepatoma PM.

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.

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.

While carcinogenesis is known to induce various alterations in epithelial cell polarity, little information is available about the fate of plasma membrane polarity during the neoplastic process. Using three monoclonal antibody-defined antigens as markers of the three plasma membrane domains of rat hepatocytes, the sinusoidal, the lateral, and the canalicular, we demonstrated by immunohistochemical techniques that changes in hepatocyte plasma membrane polarity occur at every stage of 3'-methyldimethylaminoazobenzene-induced hepatocarcinogenesis. At the preneoplastic stage, the division of hepatocyte plasma membrane into three distinct domains was retained despite rearrangements in hepatocytic architecture, characterized by the disorganization of hepatocytic plates and the formation of pseudoacinar structures. The most striking change was the distribution of the canalicular-associated antigen over the entire plasma membrane in disorganized plates. At the neoplastic stage, changes in plasma membrane polarity depended on the degree of morphological differentiation of neoplastic cells. Poorly differentiated cells inconstantly expressed the monoclonal antibody-defined antigens and showed no evidence of plasma membrane polarization. Differentiated cells constantly expressed the three antigens, and their plasma membrane was divided into antigenically distinct domains. The changes in hepatocyte plasma membrane polarity demonstrated in situ during 3'-methyldimethylaminoazobenzene hepatocarcinogenesis may therefore be compared with situations known to occur in vitro, in cultured epithelial cells presenting varying degrees of polarization. Our observations suggest that these alterations are of relevance to the in vivo biology of cancer.

Donryu strain albino rats were maintained on a diet containing 0.06% 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) for nine successive generations. Some rats in the fourth to eighth generations showed marked resistance to the carcinogenic action of 3'-Me-DAB. In the liver where we found tumors, their size and number are smaller than in the corresponding original strain of rats fed on a diet containing 3'-Me-DAB. No significant differences were found in the total cytochrome P-450 contents or epoxide hydrolase activities of the livers of the resistant variant and the original strain, but the benzo(a)pyrene hydroxylase activity which is mainly attributed to cytochrome P-448 and glutathione S-transferase activity of the resistant variant were lower. The inductions of hepatic cytochrome P-488 and benzo(a)pyrene hydroxylase on administration of polychlorinated biphenyls or 3-methylcholanthrene were also lower in the resistant rats. In the mutagenicity test on Salmonella typhimurium TA 98 the liver 9000 X g supernatant fraction from 3'-Me-DAB-resistant F7 rats did not fully induce the mutagenicities of 3'-Me-DAB and several other carcinogens. Thus the resistance of F7 rats to the chemical carcinogen may be related to the lower activities of some drug-metabolizing enzymes and the poor inducibility of cytochrome P-448 in their liver, although selection of resistant rats should be continued for further generations before coming to a definite conclusion on biochemical basis of apparent resistance to 3'-Me-DAB.

The location of binding sites for 3'-methyl-p-dimethylaminoazobenzene (3'-Me-DAB) or metabolites on components of rat liver cells and hepatoma cells in tumors induced by this carcinogen was determined at 2 stages during the induction of tumors in rats: (a) in normal liver immediately following the application of a massive dose of the azocarcinogen by intragastric feeding, and (b) in liver and tumor after hepatomas had developed following repeated exposures to the carcinogen by s.c. injections. Bound 3'-Me-DAB or metabolites were detected by the use of rabbit antisera directed against either p-azoazobenzene or p'-azo-p-dimethylaminoazobenzene in an indirect fluorescent antibody technique. Soon after massive intragastric doses of 3'-Me-DAB, the staining observed when the anti-p-azoazobenzene antiserum was used was principally on cytoplasmic components of liver cells with some staining of the intranuclear components. When the second antiserum, anti-p'-azo-p-dimethylaminoazobenzene antiserum, was used, the most intense fluorescent staining was on the nuclear membranes, although there was some cytoplasmic and intranuclear staining as well.

Activities of the adenine purine nucleotide cycle enzymes, i.e., adenylosuccinate (SAMP) synthetase, SAMP lyase, and adenosine 5'-monophosphate deaminse, were determined in hepatic tissue of rats fed and/or given injections of 3'-methyl-4-dimethylaminoazobenzene, 4'-methyl-4-dimethylaminoazobenzene, thioacetamide, ethionine, or 2-acetylaminofluorene. SAMP lyase activity showed an early increase in all regimens containing hepatocarcinogens. Adenosine 5'-monophosphate deaminse showed increases with 3'-methyl-4-dimethylaminoazobenzene and thioacetamide but not with ethionine or 2-acetylaminofluorene. SAMP synthetase either was nonresponsive or else showed inhibition to the carcinogens. Increase in SAMP lyase activity was noted as early as 48 to 72 hr following i.p. injections of these carcinogens. The response of SAMP lyase was not duplicated by analogs of carcinogens such as 4'-methyl-4-dimethylaminoazobenzene or methionine. These data imply interaction of active carcinogens with SAMP lyase and to some extent adenosine 5'-monophosphate deaminse or to some mechanism responsible for their synthesis and/or release. This interaction may be a significant component of the initiation phase of carcinogenesis.

RNA sulfurtransferase activity has been detected in rat liver and in hepatomas from rats fed a diet containing 0.06% 3'-methyl-4-dimethylaminoazobenzene for 14 to 18 weeks. The reaction measured was the transfer of sulfur from cysteine to acceptor sites in Escherichia coli B transfer RNA (tRNA). Specific activities of the enzymes in liver and hepatoma supernatant fractions were similar, as were the rates and extents of sulfur transfer to tRNA. DEAE-cellulose chromatography of digests of the [35S]tRNA reaction products revealed 3 peaks associated with nucleotide material, the amounts of these peaks differing in tRNA from liver and hepatoma systems. This may suggest differences in specific sulfurtransferases in these tissues.

A highly purified phenylalanine transfer RNA (tRNAPhe) was isolated from a normal rat liver and from livers of male and female rats fed a semisynthetic diet containing 0.06% 3'-methyl-4-dimethylaminoazobenzene for 3 weeks. Absorption spectral analysis on tRNAPhe from normal and carcinogen-fed rats indicated an unusual absorption above 335 nm by tRNAPhe isolated from the latter group. Ribonuclease T1 digestion followed by chromatography on diethylaminoethyl cellulose columns indicated the existence of a major peak of covalently bound oligonucleotide-azo complex. Microcrystalline cellulose thin-layer chromatography resolved the peak into one major and four minor components, all with similar absorption spectra. Enzymatic digestion of the major peak obtained from diethylaminoethyl cellulose chromatography to ribonucleosides, followed by chromatography on cellulose thin layer, resulted in one major and three minor components. A higher uridine:cytidine base ratio was also observed in the tRNAPhe isolated from the carcinogenfed animals. These findings suggest that certain transfer RNA's may be major targets for this azo carcinogen.

The DNA content and ploidy state of cells isolated from the livers of rats exposed to the carcinogen 3'-methyl-4-dimethylaminoazobenzene for 10 and 20 weeks, as determined by flow cytometry, were correlated with the development of oval cells in the livers of treated animals and with serum levels of the oncoprotein alpha-fetoprotein (AFP). The study revealed that there was initially a steady rise in the AFP levels found in the carcinogen treated rats. Associated with this increase was a change in the ploidy pattern of the liver from an approximately equal mixture of tetraploid and diploid cells to a predominantly diploid state. Histologically, there was an increase in the number of oval cells during carcinogen treatment, and when stained immunohistochemically for AFP, these cells were positive. We conclude that the changing state of the diploid and tetraploid cell populations is due to the proliferation of oval cells and that these cells are responsible for the initial increase of serum AFP. The maintenance of the diploid population of cells at later periods of the study is a reflection of the persistence of a new cell type, possibly derived from oval cells. The effect of 3'-methyl-4-dimethylaminoazobenzene was not reversed if the animals were withdrawn from the diet at 10 weeks. In addition, in the cases of hepatocellular carcinoma that were found, a population of cells was detected by flow cytometry that contained altered DNA.