Production of Formaldehyde and DNA−Adriamycin or DNA−Daunomycin Adducts, Initiated through Redox Chemistry of Dithiothreitol/Iron, Xanthine Oxidase/NADH/Iron, or Glutathione/Iron

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Abstract
The reaction of the antitumor drugs adriamycin and daunomycin with the self-complementary DNA oligonucleotide (GC)4 to generate DNA−drug adducts was investigated as a function of redox reaction conditions. The redox systems dithiothreitol (DTT)/Fe(III) and xanthine oxidase/NADH both gave the same distribution of four DNA−anthracycline adducts. In each of these adducts the anthracycline is bonded via a methylene linkage between the 3‘-amino group of the drug and the 2-amino group of a deoxyguanosine of the DNA. The methylene linkage results from reaction of the drug and DNA with in situ-generated formaldehyde via Schiff base chemistry [Taatjes, D. J., Gaudiano, G., Resing, K., and Koch, T. H. (1997) J. Med. Chem. 40, 1276−1286]. Formaldehyde production is promoted by iron, inhibited by metal-chelating agents, and does not require drug. Iron enhances formaldehyde production by a factor of 30, EDTA inhibits its formation by a factor of 2, and Desferal inhibits its formation by a factor of more than 20. Hydrogen peroxide accumulates in significant quantities only with xanthine oxidase/NADH in the presence of Desferal. The results are explained in terms of Fenton oxidation of Tris buffer to formaldehyde. Biological reagents also cause DNA−drug adduct formation; reduction of ferric ion with glutathione in phosphate buffer in the presence of spermine produced the same DNA−drug adducts. The observations are discussed in terms of cytotoxicity resulting from iron chelated to adriamycin catalyzing in vivo production of formaldehyde which links adriamycin to DNA and tumor cell resistance resulting from factors which decrease formaldehyde.