Quantitative and qualitative aspects of the binding of N-hydroxy-2-acetylaminofluorene to hepatic chromatin fractions

The binding of a chemical carcinogen to components of hepatic chromatin in male rats was examined. After a single injection of N-[³H]hydroxy-2-acetylaminofluorene ([³H]OH-AAF) covalent binding to chromatin RNA, protein, and DNA occurs. The amount of carcinogen bound to RNA was approximately 5 times greater than to DNA, and 10 times that of the protein. However, loss of carcinogen from RNA with time was rapid, whereas a persistent binding to DNA equal to 15% of the initial values was observed. To localize the initial and persistent DNA-bound carcinogen, the genome was fractionated using two different chromatin fractionation procedures. The procedures used yielded 3 chromatin fractions based on physical characteristics, degree of association with nascent RNA and in vitro template capacity. Based on those parameters, these chromatin fractions have been tentatively classified as template expressed euchromatin, a repressed heterochromatin, and a highly condensed pelleted heterochromatin. With both the glycerol gradient chromatin fractionation procedure and the selective MgCl₂ chromatin precipitation prooedure, the initial (2h) binding of carcinogen was greatest on the euchromatin DNA. Loss of carcinogen from the DNA, however, was also significantly faster from the euchromatin when compared to the heterochromatin and the pelleted heterochromatin. By 10 days after a single injection of the carcinogen, the largest amount of bound fluorene residues was located on the pelleted heterochromatin DNA, an apparently repressed portion of the genome, while less than 5% of the initial values were found on either the eu- or heterochromatin. When the rats were fed a 2-acetylaminofluorene-containing diet, loss of carcinogen from the pelleted heterochromatin DNA was enhanced, while loss from the euchromatin DNA was reduced. The covalent nature of the carcinogen modification of DNA was confirmed by thin-layer chromatography (TLC). These studies also demonstrated 2 separate carcinogen-purine base adducts which were identified as N-(guanin-8-yl)-N-AF and 3-(guanin-N²-yl)-N-AAF based on either co-chromatography with an authentic standard or on published R_f-values, respectively. The pelleted heterochromatin DNA had a significantly greater proportion of the 3-guanine-N² adduct when compared to DNA from either the eu- or heterochromatin.