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DNA Adducts of the Antitumor Agent Diaziquone¹

Department of Preventive Medicine and Environmental Health [R. C. G., A. G., K. E.] and Graduate Center for Toxicology [R. C. G.], University of Kentucky, Lexington, Kentucky 40506; Environmental Health Research and Testing, Research Triangle Park, North Carolina 27709 [S. C. A., G. R. L.]; and Carcinogenesis and Metabolism Branch, US Environmental Protection Agency, Research Triangle Park, North Carolina 27711 [S. N.]

We have studied adduct formation of the antineoplastic agent diaziquone (AZQ; NSC 182986) with DNA and nucleotides in vitro. The aziridine 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 ³²P-postlabeling assay involving purification of the nuclease P₁-enriched labeled adducts by high-salt C₁₈ 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 concentrations of AZQ and DNA (1 µg/µl each), peak binding was observed in about 2 h [1926 ± 378 (SD) fmol/µg 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/µg 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 monoaminoalcohol 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 ³²P-postlabeling assay seems capable of detecting relatively polar adducts such as those formed with AZQ at a level of one adduct/10⁹ nucleotides.

¹ This research was supported in part by the US Environmental Protection Agency Cooperative Research Agreements CR-813840 and CR-816185. Presented in part at the 31st Annual Meeting of the American Association for Cancer Research, Inc. (33).

² To whom requests for reprints should be addressed, at Preventive Medicine and Graduate Center for Toxicology, 207 Funkhouser Building, University of Kentucky, Lexington, KY 40506-0054.

Received 4/29/91. Accepted 7/19/91.