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

Biphasic removal of DNA adducts has previously been demonstrated by ra dioimmunoassay in whole liver DNA from rats chronically fed 2-acetylam inofluorene for 28 days. In the present study, removal of N-(deoxyguan osin-8-yl)-2-aminofluorene was observed in situ by microfluorometry. F rozen 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. Hoechs t dye was used to identify DNA in nuclei. During the 28 days on the co ntrol diet, after 28 days of feeding 2-acetylaminofluorene, the DNA ad duct concentrations of parenchymal liver cells were reduced by 85%, as compared to animals fed only the carcinogen for 28 days. Periportal h epatocytes exhibited biphasic (fast and slow) adduct removal. Only fas t adduct removal was demonstrated in midzonal and centrilobular hepato cytes, since the adduct levels were below the detectable range in thes e regions after 7 days on the control diet. After 28 days on the contr ol diet, N-(deoxyguanosin-8-yl)-2-aminofluorene was detected in simila r to 50% of periportal hepatocytes. These results are compatible with the previously observed biphasic removal profile determined by radioim munoassay of whole liver DNA adducts and indicate that periportal hepa tocytes remove adducts from two distinct genomic compartments.