Reduced DNA adduct formation in replicating liver cells during continuous feeding of a chemical carcinogen

To investigate early cellular alterations in liver DNA during hepatocarcinogenesis, we have visualized replicating cells and analyzed their DNA adduct content in livers of rats continuously fed a carcinogenic level (0.02%) of 2-acetylaminofluorene for periods up to 4 weeks. One hour prior to sacrifice, cells undergoing DNA synthesis were pulse-labeled with the thymidine analogue 5-bromodeoxyuridine. Replicating cells were visualized immunohistochemically with anti-(5-bromodeoxyuridine), and identification of aminofluorene-DNA adducts in replicating nuclei was achieved by staining with an antiserum specific for N-(deoxyguanosin-8-yl)-2-aminofluorene; both stains were observed simultaneously by two-color immunofluorescence. Data were obtained for all cells, including large hepatocytes (nuclei greater than 6 microns) and small cells (nuclei less than 6 microns), such as hepatocytes sliced asymmetrically, oval cells, Kuppfer cells, and sinusoidal lining cells. Based on the size of their nuclei, the hepatocytes were the only cells that could be identified separately from the total. A distinct increase in the number of cells synthesizing DNA was observed after 25 days of 2-acetylaminofluorene feeding; replicating cells were either scattered randomly throughout the liver or clustered in discrete foci. At times up to 28 days, cells with both large and small nuclei that were synthesizing DNA showed reduced aminofluorene-DNA adduct immunofluorescence compared to nonreplicating cells. The results suggest that liver cells replicating during carcinogen exposure have altered metabolic capacities resulting in reduced aminofluorene-DNA adduct formation. It is possible that such cells constitute the progenitors of preneoplastic foci, which have a replicative advantage as compared to normal liver.