Dose response of early effects related to tumor promotion of 2-acetylaminofluorene

The genotoxic effects of 2-acetylaminofluorene (AAF) alone cannot explain the formation of rat liver tumors. It has been proposed that mitochondrial effects are associated with its tumor-promoting properties. These mitochondrial effects are thought to trigger apoptosis and regenerative proliferation, which alters the liver lobule in a cirrhosis-like manner. A situation is generated which favors the growth of initiated cells. To test this sequence of events, the dose dependence of early effects with time was studied. Male Wistar rats received 50, 100, 200, 400, or 800 ppm AAF in the diet and the following endpoints were analyzed at 2, 4, 8, and 16 weeks of feeding: apoptotic cell death, cell proliferation, GST-P-positive foci (placental form of glutathione S-transferase), and morphological alterations. Hydrolyzable hemoglobin adducts were used as a dosimeter for metabolic activation after 2 weeks of feeding. The hemoglobin adduct levels increased linearly with dose. With the conventional carcinogenic concentration of 200-ppm AAF in the diet, the number of apoptoses increased first, predominantly in the periportal area (2 weeks). Cell proliferation followed with some delay (4 weeks). This reflects regenerative tissue response and not the growth of initiated cells, because the number of enzyme-altered foci was still extremely low at that time. Foci developed only later when the morphology had changed. With 50 ppm AAF in the diet, a no-effect level had not been reached for any of the endpoints, but foci developed much more gradually than with higher doses. Unexpectedly, the proliferative response stabilized at 8 weeks with a labeling index of 12-17, with all AAF concentrations. The observed sequence of events supports the hypothesis. It is concluded that (1) The proliferation of initiated cells-defined as promotable cells-is largely determined by the cellular environment, such as morphologically altered liver. (2) The morphological alterations in rat liver result from imperfect regeneration from toxic effects. (3) Imperfect regeneration results from limitations in the possibilities to adapt to chemical stress. (4) If these limits are overwhelmed and morphology has changed to a certain extent, preneoplastic foci develop; this occurs much faster, at least, than without these changes.