Sequential observation of pathomorphologic alterations in preneoplastic lesions during the promoting stage of hepatocarcinogenesis and the development of short-term test system for hepatopromoters and hepatocarcinogens
Authors: Ito Nobuyuki , Tsuda Hiroyuki , Hasegawa Ryohei , Imaida Katsumi 
Published: September 2009

The aims of this study were 1) to observe the sequential development of hepatocellular carcinomas from preneoplastic lesions and to investigate hyperplastic (neoplastic) foci or nodules (HN) as an indicator of a preneoplastic population, and 2) to test the promoting effect of various agents and to study the dose-dependent effect of promoting agents in the induction of preneoplastic lesions in the rat liver.

1) F344 rats were injected with N-nitrosodiethylamine (DEN) and then given basal diet containing 2-acetylaminofluorene (2-AAF) or α-hexachlorocyclohexane (α-HCH). Two-thirds partial hepatectomy (PH) was performed at the end of week 3. Animals were killed periodically for quantitative analysis of HN and for study of changes in blood supply to the lesions by method using a resin. In the liver of rats treated with 2-AAF after DEN, the number and area of HN were maximal in week 10, and then the number gradually decreased to week 50 (P < 0.001), whereas the area remained almost constant. In the group given α-HCH after DEN, the number of HN decreased temporarily in week 20 and then gradually increased, whereas the area of HN increased slowly throughout the experiment. Histological examination suggested that the decrease of HN after week 10 was due to degeneration of HN with their change to a spongy or cystic appearance, and that the degeneration resulted from circulatory disturbance. The number and area of these degenerating hyperplastic nodules (DHN) increased reciprocally to the decrease of HN with time until week 30. The number of hepatocellular carcinomas was maximal at week 40.

The blood supply to the early hyperplastic nodules was mainly through the portal vein as with normal or surrounding liver tissue, but at a later stage HN and hepatocellular carcinomas were supplied with a blood mainly from the hepatic artery. Therefore, arterial blood supply seemed important for the persistence of HN and development of hepatocellular carcinoma. Most of the HN which appeared within the first 10 weeks were histochemically positive for γ-glutamyl-transpeptidase (γ-GT) activity. This experiment showed that detectable preneoplastic lesions measured as γ-GT positive foci or HN were induced by exposure to promoting agents for 6 to 10 weeks after initiation with DEN.

2) In tests of the promoting activity and dose-dependent effect of various compounds, rats were injected intraperitoneally with DEN and given the test compounds for 6 to 10 weeks and then killed. PH was performed as in experiment 1. Potent hepatocarcinogens, such as 2-AAF, 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB), ethionine and N-nitrosodimethylamine (DMN), induced a large number and area of HN or γ-GT positive foci whereas weak ones, such as α-HCH, dieldrin, hormones and bile acids evoked less response. Both potent and weak carcinogens showed a clear dose-dependent effect. A similar dose-dependent effect was also shown in the induction of hepatocellular carcinoma in a long-term experiment by continuous feeding of DMN. Non-hepatocarcinogens, such as N-ethylnitrosourea (ENU) and 3-methylcholanthrene (3-MC) induced them only in small numbers.