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

The genotoxic effects of 2-acetylaminofluorene (AAF) alone cannot explain t he formation of rat liver tumors. It has been proposed that mitochondrial e ffects are associated with its tumor-promoting properties. These mitochondr ial 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. Mal e Wistar rats received 50, 100, 200, 400, or 800 ppm AAF in the diet and th e following endpoints were analyzed at 2, 4, 8, and 16 weeks of feeding: ap optotic 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 d ose. With the conventional carcinogenic concentration of 200-ppm AAF in the diet, the number of apoptoses increased first, predominantly in the peripo rtal 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 a t that time. Foci developed only later when the morphology had changed. Wit h 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 dos es. Unexpectedly, the proliferative response stabilized at 8 weeks with a l abeling index of 12-17, with all AAF concentrations. The observed sequence of events supports the hypothesis. It is concluded that (1) The proliferati on of initiated cells-defined as promotable cells-is largely determined by the cellular environment, such as morphologically altered liver. (2) The mo rphological alterations in rat liver result from imperfect regeneration fro m toxic effects. (3) Imperfect regeneration results from limitations in the possibilities to adapt to chemical stress. (4) If these limits are overwhe lmed and morphology has changed to a certain extent, preneoplastic foci dev elop; this occurs much faster, at least, than without these changes.