Covalent Intercalate Binding to DMA in Relation to the Mutagenicity of Hydrocarbon Epoxides and N-Acetoxy-2-acetylaminofluorene1

The abilities of N-acetoxy-2-acetylaminofluorene and a series of hydrocarbon epoxides to cause the unwinding of supercoiled SV40 DNA by covalent binding were mea sured by incubating the DNA with the compound at pH 7.4 at 37° and then determining the superhelicity of the modi fied DNA by gel electrophoresis. The concentrations of the electrophiles required to unwind fully the supercoiled DNA ranged from 0.023 to :>3 mu in this ascending order: (±)-7/3,8a-dihydroxy-9a,10a-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, 1-pyrenyloxirane, 9-methyl-10-anthryloxirane, 6-chrysenyloxirane, 9-anthryloxirane, N-acetoxy-2acetylaminofluorene, 9-phenanthryloxirane, 7-benzanthryloxirane, 2-naphthyloxirane, and styrà ̈neoxide (phenyloxirane). The frame-shift mutagenicities of these com pounds toward Salmonella typhimurium TA98 ranged from 540 to 0.001 revertants/nmol and were well correlated with their abilities to cause DNA unwinding (Kendall's coefficient of rank correlation, f = 0.847, p < 0.002). The abilities of the compounds to induce missense mutations in S. typhimurium TA100 did not correlate as well with their activities in the DNA-unwinding assay (f = 0.548, p <0.1). The hydrolysis rates of the compounds under the incubation conditions used for the DNA unwinding assay were not correlated with their mutagenicities or their activities in causing DNA unwinding. Studies with N-acetoxy-2-[3H]acetylaminofluorene and (±)-[7-14C]-7j3,8a-dihydroxy-9a,10a-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene indicated that complete unwinding of the supercoiled DNA required binding of about 0.05 hydrocarbon residue/nucleotide. These values are similar to those obtained for the noncovalently bound intercalating agent ethidium bro mide. The changes in superhelicity of the SV40 DNA on treatment with these two electrophiles were confirmed by electron microscopy; no denaturation of the modified DNA was observed. These data suggest a model of covalent intercalative binding for the interaction of these com pounds with DNA.