RT Journal Article SR Electronic T1 Mutations and Decreases in Density of Transforming DNA Produced by Derivatives of the Carcinogens 2-Acetylaminofluorene and N-Methyl-4-aminoazobenzene JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 411 OP 426 VO 4 IS 5 A1 VERONICA M. MAHER A1 ELIZABETH C. MILLER A1 JAMES A. MILLER A1 WACLAW SZYBALSKI YR 1968 UL http://molpharm.aspetjournals.org/content/4/5/411.abstract AB A series of metabolites and synthetic derivatives of the carcinogens 2-acetylaminofluorene (AAF) and N-methyl-4-aminoazobenzene (MAB) was tested at pH 7.5 and room temperature for interaction with biologically active DNA isolated from Bacillus subtilis. Previous studies have indicated that AAF and MAB are converted in rat liver, via N-hydroxy metabolites, to reactive carcinogenic esters which can attack guanine residues in nucleic acids. In the present work the reactive synthetic esters N-acetoxy-AAF, N-benzoyloxy-AAF, AAF-N-sulfate, and N-benzoyloxy-MAB caused: (a) severe reductions in transforming activity, (b) up to 100-fold increases in the frequencies of mutations in transforming DNA, and (c) decreases in the buoyant density of DNA as measured in a CsCl gradient. MAB, AAF, and certain metabolites of AAF less carcinogenic than the reactive esters, viz., N-hydroxy-AAF, the glucuronide of N-hydroxy-AAF, 2-aminofluorene (AF), and N-hydroxy-AF, neither inactivated nor caused mutations or density changes in the transforming DNA. 2-Nitrosofluorene-treated transforming DNA exhibited a marginal increase in mutational frequency. The high carcinogenicities of N-acetoxy-AAF, N-benzoyloxy-AAF, and N-benzoyloxy-MAB, especially at the sites of subcutaneous injection in rats, correlate well with their high reactivities and mutagenicities for transforming DNA. Similarly, the lack of activity of the nonester derivatives in the transforming DNA system is in accord with their lack of reactivity with nucleophiles at neutrality. In DNA samples reacted with esters of N-hydroxy-AAF, the frequencies of induced mutations were directly proportional to the decreases in buoyant density. Furthermore, experiments with N-acetoxy-AAF-9-14C demonstrated that the decrease in buoyant density was also directly proportional to the number of fluorene molecules covalently bound to the DNA. The decrease in density appears to result from two opposing effects: binding of the highly buoyant AAF residues and slight denaturation of the reacted DNA. Severely inactivated DNA, after denaturation, showed CsCl and Cs2SO4 gradient sedimentation patterns compatible with the occurrence of crosslinks between the complementary strands. Bacterial strains capable of repairing lesions in DNA caused by ultraviolet radiation restored up to 50% of the transforming ability inactivated by these reactive esters. The induced mutations were spontaneously reversible and thus appeared to be caused by single base-pair changes. ACKNOWLEDGMENT This investigation was supported by grants from the National Science Foundation (B-14976), the National Cancer Institute, USPHS (CA-07175 and CRTY-5002), the Jane Coffin Childs Memorial Fund for Medical Research, and by the Alexander and Margaret Stewart Trust Fund. We would like to acknowledge the valuable suggestions and frequent help of our colleagues Dr. H. Kubinski, Dr. H. A. Lozeron, and Dr. W. C. Summers. Mr. D. Zuhse has provided expert assistance with the ultracentrifuge, and Mr. J. T. Fiandt and Mr. T. E. Noll assisted with other technical aspects of the study.