Persistent binding of a new reaction product of the carcinogen N-hydroxy-N-2-acetylaminofluorene with guanine in rat liver DNA in vivo.

Abstract

The binding of 2-acetylaminofluorenyl residues (-AAF) to rat liver DNA in vivo was studied at different periods of time after administration of N -hydroxy- N -2-acetylaminofluorene-2′-3H. Liver DNA was hydrolyzed at pH 6 with a mixture of spleen phosphodiesterase and wheat germ acid phosphatase. The enzymatic digests were analyzed by Sephadex LH-20 column chromatography. Maximum levels of bound radioactivity were found in liver DNA at 16 to 18 hr following a single injection of N -hydroxy- N -2-acetylaminofluorene-2′-3H. The major part (80%) of the bound radioactivity was identified as N -(deoxyguanosin-8-yl)-2-acetylaminofluorene (dGuo-AAF), which disappeared rapidly from DNA with a biological half-life of approximately 7 days. A second product, however, constituting 20% of the bound radioactivity, remained associated with DNA for periods of up to 8 weeks after injection. Unlike dGuo-AAF, the minor product was not deacetylated by the action of 0.1 n NaOH or 0.1 n HCl at 75° for 2 hr. The persistent -AAF residue was not detected in rRNA and tRNA from rat liver. A minor product with chemical and chromatographic properties similar to those of the persistent -AAF moiety in vivo was isolated from 14C-labeled DNA, which had been reacted with N -acetoxy- N -2-acetylaminofluorene-2′-3H in vitro . The 3H:14C ratio of this product was identical to the theoretical 3H:14C ratio, which was calculated for the reaction product dGuo-AAF, thus indicating that the persistent -AAF moiety in DNA is also bound to guanine. Persistent binding of radioactivity to rat liver DNA in vivo was also observed following a single injection of 2-acetylaminofluorene-9-14C-2′-3H. Previous reports that 70% of the total carcinogen bound to DNA at 24 hr had lost the N -acetyl group were confirmed. Approximately 20% of bound -AAF at 24 hr (5% of total carcinogen) remained associated with DNA at 4 weeks after injection. The 3H:14C ratio of DNA at 4 weeks was identical to the 3H:14C ratio of the isolated reaction product dGuo-AAF, indicating that there are no persistent 2-aminofluorene residues. Administration of 2-acetylaminofluorene-9-14C-2′-3H resulted in 53% exchange of N -acetyl groups, but injection of N -hydroxy- N -2-acetylaminofluorene-9-14C-2′-3H did not result in significant exchange of the N -acetyl group.