DNA adduct formation and removal in hepatic chromatin fractions from rats chronically fed 2-acetylaminofluorene

Continuous dietary administration of the hepatocarcinogen 2 acetylaminofluorene (AAF) to rats produces a gradual increase in hepatic DNA adducts until a plateau is reached after ∽2 weeks. The rate of DNA adduct formation remains constant through 1 month of AAF feeding, while adduct removal profiles are biphasic during both carcinogen feeding and subsequent time on control diet. In the present experiments, we tested the hypothesis that biphasic adduct removal is due to differential repair kinetics taking place in different chromatin fractions. Rats were fed 0.02% AAF for thnes up to 30 days and control diet for a subsequent 28 days. HPLC analysis of nuclear DNA indicated that the deacetylated adduct, N-(deoxyguanosin-8-yI)-2-aminofluorene, comprised ∽90% of the total C8-substituted deoxyguanosine adducts after 3 days of feeding and >98% after 20 days. The nuclear DNA was partitioned into endogenous nuclease sensitive (∽2%), low salt soluble (∽70%), high salt soluble (∽20%) and nuclear matrix (∽8%) fractions. During 28 days of AAF feeding, each fraction showed a profile of adduct formation similar to that observed in whole nuclei; however, the adduct concentration in nuclear matrix-associated DNA was consistently less than that in the other fractions. In rats fed AAF for 28 days followed by control diet, adduct removal in each of the fractions showed biphasic kinetics that were similar to those observed in nuclear DNA. When rats were fed AAF for 7 days, however, adduct removal kinetics could be best described by a single first-order rate constant. These data indicate that biphasic adduct removal may be due to the presence of particular nucleotide sequences that are common to all fractions and are relatively resistant to adduct formation and removal. The low concentration of adducts found in the nuclear matrix may be due to a decreased rate of adduct formation in this region and/or the proximity of membrane-bound β-polymerases that are responsible for repair.