Quantitative Histochemical and Autoradiographic Studies of Hepatocarcinogenesis in Rats Fed 2-Acetylaminofluorene followed by Phenobarbital¹

Combined histochemical and autoradiographic techniques with serial cryostat sections have been developed for determining the number, type, and nuclear labeling indices after in vivo [³H]thymidine injection of hyperplastic hepatocellular islands, nodules, and carcinomas induced by feeding 2-acetylaminofluorene followed by phenobarbital to rats. A total of 195 phenotypically altered islands and 5 hepatocellular carcinomas was characterized in the livers of 6 rats that had been fed 0.02% 2-acetylaminofluorene for 4 weeks followed by 0.05% phenobarbital for 39 weeks. The islands were classified into one of seven different categories on the basis of abnormalities in content of one, two, or three different enzyme markers. These alterations, selected on the basis of earlier investigations, were (a) presence of canalicular ∮-glutamyl transpeptidase; (b) absence of canalicular adenosine triphosphatase; and (c) absence of cytoplasmic glucose-6-phosphatase. Eighty-nine % of the islands were GGT+, 71% were ATP-, but only 35% were G6P-. Islands with only the single marker change, GGT+, had mean labeling indices that were 4 times greater (2.2%) than were unaltered background hepatocytes. Loss of canalicular adenosine triphosphatase and cytoplasmic glucose-6-phosphatase in islands that were GGT+ was associated with a further growth advantage since the mean labeling index of islands with the three abnormalities was 4.4%. Furthermore, in this study, the loss of glucose-6-phosphatase served as the single best marker for islands with the highest labeling indices, since 90% of all G6P- islands were also ATP- and GGT+. All hepatocellular carcinomas were highly differentiated, and three of them showed areas of cystic degeneration. While the mean labeling index of all macroscopic nodules and carcinomas was 11%, the two solid hepatocellular carcinomas were found to have labeling indices that were 5 to 6 times greater than were the hyperplastic nodules from which they appeared to arise.