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Persistent Binding of a New Reaction Product of the Carcinogen N-Hydroxy-N-2-Acetylaminofluorene with Guanine in Rat Liver DNA in Vivo¹

Department of Biochemistry, Antoni van Leeuwenhoek-Huis, The Netherlands Cancer Institute, Amsterdam, The Netherlands

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.

¹ This is Paper 3 of the series entitled "On the Mechanism of Action of Carcinogenic Aromatic Amines."

Received 4/12/72. Accepted 6/ 8/72.

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