DNA Adducts of the Antitumor Agent Diaziquone1

We have studied adduct formation of the antineoplastic agent diaziquone (AZQ; NSC 182986) with DNA and nucleotides in vitro. The a/iridine moieties of AZQ can be expected to interact covalently with DNA which, in turn, presumably elicits the antitumor activity. We analyzed AZQ-DNA adducts by a modified "P-postlabeling assay in volving purification of the nuclease P,-enriched labeled adducts by highsalt C,»reversed-phase thin-layer chromatography and separation of the eluted adducts on a polyethyleneimine-cellulose layer using non-urea salt solutions. Modification of calf thymus DNA with AZQ produced two major (22% and 40%) and at least eight minor adducts. At equal concen trations of AZQ and DNA (1 MÃ-Ã-/V'l each), peak binding was observed in about 2 h (1926 ±378 (SD) fmol/jig of DNA| with the binding levels remaining practically unchanged through 4 h. However, incubation for 24 h resulted in over 40% decline, indicating adduct instability. AZQ was found to be highly reactive in vitro as evidenced by its substantial binding (49 ±14 fmol/fig of DNA) even at a DNA:AZQ ratio of 100:1. When incubated with mononucleotides, AZQ reacted extensively with adenine, guanine, and cytosine but only slightly with thymine. Cochromatography of the modified DNA and nucleotides revealed that one of the major adducts and several minor adducts were guanine derived. The aziridine rings of AZQ were found to be the main reactive sites as its inonoaminoalcohol derivative showed as much DNA reactivity as did the parent compound, but no activity was observed when both aziridine groups were hydrolyzed to diaminoalcohols. The improved "P-postlabeling assay seems capable of detecting relatively polar adducts such as those formed with AZQ at a level of one adduci/1 (C1nucleotides.