IMMUNOHISTOCHEMICAL AND MICROFLUOROMETRIC DETERMINATION OF HEPATIC DNA ADDUCT REMOVAL IN RATS FED 2-ACETYLAMINOFLUORENE

Biphasic removal of DNA adducts has previously been demonstrated by radioimmunoassay in whole liver DNA from rats chronically fed 2-acetylaminofluorene for 28 days. In the present study, removal of N-(deoxyguanosin-8-yl)-2-aminofluorene was observed in situ by microfluorometry. Frozen liver sections from animals fed 0.02% 2-acetylaminofluorene for 28 days, followed by a control diet for 3, 7, 14, 21 and 28 days, were examined immunohistochemically for localization of N-(deoxyguanosin-8-yl)-2-aminofluorene with fluorescein-conjugated secondary antiserum. In addition, bile ducts and oval cells were stained with antibodies to keratins using Texas red-labelled indirect immunofluorescence. Hoechst dye was used to identify DNA in nuclei. During the 28 days on the control diet, after 28 days of feeding 2-acetylaminofluorene, the DNA adduct concentrations of parenchymal liver cells were reduced by 85%, as compared to animals fed only the carcinogen for 28 days. Periportal hepatocytes exhibited biphasic (fast and slow) adduct removal. Only fast adduct removal was demonstrated in midzonal and centrilobular hepatocytes, since the adduct levels were below the detectable range in these regions after 7 days on the control diet. After 28 days on the control diet, N-(deoxyguanosin-8-yl)-2-aminofluorene was detected in similar to 50% of periportal hepatocytes. These results are compatible with the previously observed biphasic removal profile determined by radioimmunoassay of whole liver DNA adducts and indicate that periportal hepatocytes remove adducts from two distinct genomic compartments.