Dose-Dependent Induction of Preneoplastic Lesions by the Tobacco-Specific Nitrosamine Carcinogen Nnk in the in Ovo Carcinogenicity Assessment (ioca) Assay

The potential of the carcinogenic tobacco specific nitrosamine 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-1-butanone (NNK) to induce preneoplastic hepatocellular altered foci (HAF) was tested in the in ovo carcinogenicity assessment (IOCA) assay. Single doses of NNK over a dose range from 0.1 mg to 6 mg were injected into fertilized turkey eggs prior to incubation for 24 days. The livers were investigated by histological, histochemical and morphometric methods. Mortality was increased for eggs exposed to 6 mg. In this group, the whole livers were severely altered, showing pronounced changes of nucleus size and signs of cell death. At the dose of 2 mg various types of foci of altered hepatocytes (HAF) were observed. Basophilic cell foci of the solid or tubular type were most frequent. The NNK-induced HAF were very similar to the preneoplastic lesions that occur in the livers of mammals during hepatocarcinogenesis which are regarded as early indicators of carcinogenesis. The similarity to the HAF in rodents included histochemically detectable alterations like decreased activities of glucose-6-phosphatase, adenosine triphosphatase and glycogen phosphorylase. At doses of 1 mg or below, no HAF were detected. At all dose levels an increased occurrence of enlarged hepatocytes with enlarged nuclei and prominent nucleoli (karyomegalic hepatocytes) were observed. The increase in karyomegalic hepatocytes was also statistically significant at the low dose of 0.1 mg/kg NNK but the dose–effect curve for their induction was clearly non-linear. Induction of HAF and karyomegalic hepatocytes in ovo is a simple (one dose), rapid (24 days) and inexpensive (no animal purchase or housing) experimental approach for studies on chemically induced hepatocarcinogenesis.