Abstract

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.