32P-Post-labelling analysis of nucleobases involved in the formation of DNA adducts by antitumor 1-nitroacridines

Abstract

Adducts generated in vitro by the reaction of 1-nitroacridines with poly(dN)s in the presence of dithiothreitol were used to identify a kind of nucleic base involved in the formation of individual adducts. The patterns of chromatographic spots corresponding to modified nucleotides obtained by 32P-post-labelling assay for synthetic homopolymers of four deoxyribonucleotides were compared with the fingerprints detected in the case of calf thymus DNA reacted with 1-nitroacridines under conditions in which the formation of identical DNA adducts as in cellular models was demonstrated in earlier investigations. Both compounds studied (Ledakrin and C-857) turned out to bind covalently only with purine nucleotides. Ledakrin formed with dG four and C-857 five different adducts. All of them were also detected in ctDNA. The incubation with poly(dA) resulted in four Ledakrin-dA species, two of which were found in ctDNA, and in two C-857-dA adducts that were not, however, observed in DNA containing samples. Modification of purines accounted for all adducts observed in ctDNA. For both compounds studied, the level of total binding to poly(dA) was about one order of magnitude lower than to poly(dG) for which it was comparable with the extent of ctDNA modification. This indicates that dG represents a preferential site of covalent binding of 1-nitroacridines to DNA.