Cell proliferation and tumour promotion by ethinyl estradiol in rat hepatocarcinogenesis.

A two-stage model of hepatocarcinogenesis is used to study the effect of exposure time to ethinyl estradiol (EE) on promotion of preneoplastic lesions in rat liver induced by diethylnitrosamine (DEN). Young male and female Sprague-Dawley rats initiated by a single dose of DEN (100 mg/kg) were subjected to different times of EE administration incorporated into the diet at 10 p.p.m. (0.5 mg/kg x day). Animals were killed 1 year after initiation. Whereas macroscopic tumours were rarely seen in animals with short exposure (3 or 4 months) or in only-initiated controls, all the animals under a long period of administration (8 months) showed macroscopic tumours. Morphometric studies on glutathione-S-transferase (GST) positive preneoplastic lesions revealed an increase in the mean size of foci and nodules corresponding to 8 months of treatment, whereas no changes were observed between animals with short exposure and only-initiated controls. No differences were seen in the incidence of these lesions between any of the protocols. In addition to an acute hyperplastic effect on non-initiated liver described earlier, our preliminary results suggest cytotoxicity and an enhancement of the liver cell turnover after several months of continuous EE administration. These results taken together suggest that promotion of hepatocarcinogenesis by EE largely depends on the time of exposure to the compound and that chronic effects on the liver cell turnover may play an important role in its ability to promote hepatocarcinogenesis.

Cell phenotype instability in preneoplastic foci of rat liver.

Carcinogenesis is a multi-step process in which genetic--phenotypic instability and sequential selection of preneoplastic cells for increased growth capacity and other neoplastic characteristics are essential phenomena. During chemical carcinogenesis in rat liver, the development of enzyme-deficient foci, their clonal origin and their relationship to tumour formation are known. We report the results of four carcinogenesis protocols consisting of one or two cycles of diethylnitrosamine and phenobarbital. Histochemistry of three enzymes on serial sections has revealed seven different kinds of homogeneous liver foci, resulting from simple and combined enzyme deficiencies, and also heterogeneous foci showing smaller foci inside. We consider such secondary foci as subclones originating from cells already modified that have developed an additional phenotypic change. Some of such foci develop after the first cycle if the promotion phase is as long as 57 weeks. Comparing the number of foci per surface area of liver section with the number of secondary foci per surface area of focus section, it seems clear that cells already modified are less stable than other hepatocytes, showing a higher tendency to develop secondary changes.

Proliferative response of putative preneoplastic hepatocytes to triiodothyronine, aminoacids, glucagon and heparine.

Proliferative advantage of putative preneoplastic hepatocytes measured as the ratio of tritiated thymidine incorporation into cells of hyperplastic nodules with respect to the incorporation in hepatocytes of extranodular liver has been found to be about 2 in rats treated according to 4 carcinogenesis protocols consisting in one or two cycles of diethylnitrosamine and phenobarbital administration. The proliferative response of hepatocytes in hyperplastic nodules to the intravenous infusion of triiodothyronine, amino-acids, glucagon, and heparine (TAGH) has been found higher or similar--except in one case--than the response of surrounding liver but the proliferative advantage of preneoplastic hepatocytes is lower after TAGH stimulation than in basal conditions in all cases.

Enzyme pattern and growth rate of liver preneoplastic clones during carcinogenesis by diethylnitrosamine.

Enzyme biochemical and histochemical assays during chemical carcinogenesis in rat liver have revealed that several adult enzyme activities are lost and some fetal enzyme activities are re-expressed in the hyperplastic foci as well as in the developed hepatomas. How these enzyme alterations are acquired and to what extent these changes are specifically related to the growth alterations leading to neoplastic development are decisive questions. Using a carcinogenesis protocol that combines a single dose of diethylnitrosamine and phenobarbital given continuously as the promoting agent and by assaying serial liver sections for glucose-6-phosphatase, adenosine-5'-triphosphatase and 5'-nucleotidase, we have identified the three-enzyme pattern of 1,746 islands and measured their section areas. We found a clear trend that clones with more deviated enzyme pattern grow faster than less deviated ones.

[Enzymatic phenotype of preneoplastic islands induced in the rat liver by diethylnitrosamine]. / Fenotipo enzimático de islotes preneoplásicos inducidos en hígado de rata con dietilnitrosamina.

Diethylnitrosamine was injected to Sprague-Dawley rats after a partial hepatectomy, and enzyme histochemistry analysis was made after 35 weeks in the preneoplastic liver. The areas and volumes of the induced enzyme-deficient islands were calculated for glucose-6-triphosphatase, adenosine triphosphatase, and 5'-nucleotidase. The preneoplastic enzyme-deficient zones showed also an increased incorporation of tritiated thymidine.