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We examined the modifying effect of hemicalcium ascorbate (Ca-Asc), and its lipophilic derivatives, 2-O-octadecylascorbic acid (CV-3611) and ascorbyl palmitate (AscP), on hepatocarcinogenesis by 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) in ODS rats (a mutant unable to synthesize ascorbic acid). Male 14-week-old ODS rats were given a modified AIN-A diet or the diet containing 0.06% 3'-Me-DAB, and drinking water containing 0.1% ascorbic acid. Rats were divided into the following eight groups: Group 1, no treatment (basal diet alone); Group 2, Ca-Asc; Group 3, CV-3611; Group 4, AscP;Group 5, 3'-Me-DAB; Group 6, 3'-Me-DAB + Ca-Asc; Group 7, 3'-Me-DAB + CV-3611; and Group 8, 3'-Me-DAB + AscP. Ca-Asc (2 g/kg), CV-3611 (0.2 g/kg), and AscP (0.6 g/kg) was administered once every day by gavage. 3'-Me-DAB was given in the basal diet. After 17 weeks, animals were killed by exsanguination, and the liver was weighed and processed for histological examination. Treatment by CV-3611 exerted a marked inhibitory effect on the development of 3'-Me-DAB-induced hepatocellular carcinomas (HCC) as measured by multiplicity. Although less effective than CV-3611, Ca-Asc and AscP also showed inhibitory effect. We have also studied the correlation of erythrocyte (RBC) polyamine levels and HCC development. RBC polyamine levels were inhibited by Ca-Asc and its derivatives, indicating it may be a marker of hepatocarcinogenesis.

Several compounds were evaluated in nonhuman primates for their potential to induce neoplasms, especially hepatocellular carcinoma (HCC). The compounds can be classified into three groups: food contaminants, model rodent carcinogens, and nitrosamines. All three compounds in the food contaminants group, namely, aflatoxin B1, sterigmatocystin, and methylazoxymethanol acetate, induced HCC. None of the model rodent carcinogens tested consistently induced HCC in rhesus and cynomolgus monkeys. Three of four nitrosamines evaluated induced HCC in rhesus and cynomolgus monkeys. One nitrosamine, diethylnitrosamine, is a predictable and potent inducer of HCC and is useful for establishment of a nonhuman primate model for numerous oncologic studies.

A new hypothesis leading to a new model of liver carcinogenesis is described; it is based on the acquisition by carcinogen-altered hepatocytes during initiation of a new functional handle--resistance to the cytotoxicity of a carcinogen--and on the ability of such cells to proliferate in an environment that prevents proliferation of normal hepatocytes. The creation of such a differential environment now enables a quantitative analysis for initiation, the beginning synchronization of the putative premalignant hepatocytes for about 15 cell cycles, the study of the pattern of growth of such resistant cells to form nodules that have some resemblance to the organizational pattern of fetal liver, the analysis of the appearance of distinctive positive and negative markers for these cells, and the further investigation of the development of liver cancer from such cells. The remarkable similarity in overall pattern betweeen the development of cancer in the skin and in the liver with chemicals and the possible role of both somatic mutation and neodifferentiation in carcinogenesis are briefly discussed.

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.

Ascites hepatoma AH-66 and 3'-Me-DAB-induced hepatoma of rats contain highly active N-acetylglucosaminyltransferase III (GnT-III) which catalyzes the addition of N-acetylglucosamine through a beta 1-4 linkage (bisecting N-acetylglucosamine) to the beta-linked mannose of the trimannosyl core of asparagine-linked sugar chains, whereas normal rat liver contains very little. The high activity was also detected in fetal rat liver, newborn rat liver, hyperplastic nodules and various transplantable hepatomas.

Changes of glycylproline dipeptidyl aminopeptidase (GPDA) and gamma-glutamyl transpeptidase (gamma-GTP) activities were compared in the serum and liver tissue of rats with hepatic cancer induced by 3'-methyl DAB. Serum glycylproline dipeptidyl aminopeptidase activity in rats with the azo dye-induced hepatic cancer was significantly higher than that in healthy rats, but the increase was not so extensive compared with that of gamma-glutamyl transpeptidase. The specific activity of glycylproline dipeptidyl aminopeptidase was decrease in the microsomal fraction and increased in the supernatant fraction of hepatic cancer tissue, whereas that of gamma-glutamyl transpeptidase was increased in both microsomal and supernatant fractions. These results suggest that the mechanisms, whereby serum activities of these two enzymes were increased in rats with hepatic cancer, were different from each other.

The feeding of carcinogenic 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) in the early stages results in a change in the protein composition of the nuclear ribonucleoprotein particles of the rat liver. These particles are associated with newly synthesized RNA and it is assumed that they are involved in the processing and in the transport of this RNA. After 6 weeks of feeding of this azocarcinogen, the amount of one of the main polypeptides (apparent molecular weight 42 000) is decreased and after 10 weeks of feeding the particles are devoid of this polypeptide completely. Feeding of the non-carcinogenic p-aminoazobenzene (AB) is without any effect. The loss of this polypeptide is not characteristic for the malignant transformation. In the nuclear ribonucleoprotein particles isolated from hepatoma which has been induced by 3'-MeDAB this polypeptide is present in even higher proportion to other polypeptides than it is in particles isolated from liver cells of control animals. The 3'-MeDAB binds to the proteins of the liver nuclear ribonucleoprotein particles and interferes with the RNA processing. It is proposed that the changes in the composition of the protein moiety of the particles reflect changes in the population of liver cells leading finally to the selection of hepatoma cells which are resistant to the toxic effect of 3'-MeDAB on RNA processing.

When rats are subjected to chemical hepatocarcinogenesis according to the protocol of D. Solt and E. Farber ((1976) Nature (London) 263, 701-703), the liver exhibits elevated levels of tyrosine protein kinase activity as early as 3 weeks after the injection of diethylnitrosoamine. A more striking elevation in tyrosine protein kinase activity is noted in rat hepatomas induced by administration of chemical carcinogens, in particular that of 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB). Tyrosine protein kinase solubilized from the particulate fraction of 3'-Me-DAB-induced hepatoma has a molecular weight identical to that of p60v-src, cross-reacts with p60v-src immunologically, phosphorylates the heavy chain of anti-p60v-src IgG, and probably belongs to a family of p60c-src. The tyrosine protein kinase from the particulate fraction of normal rat liver is indistinguishable from the hepatoma kinase in these properties; thus it apparently differs only in the level of activity. Whether the liver and hepatoma kinases differ merely quantitatively or whether they differ even qualitatively, however, remains to be elucidated.