REDOX PATHWAY LEADING TO THE ALKYLATION OF DNA BY THE ANTHRACYCLINE, ANTITUMOR DRUGS ADRIAMYCIN AND DAUNOMYCIN

Reaction of the anthracycline, antitumor drugs adriamycin and daunomyc in with the self-complementary DNA oligonucleotide GCGCGCGC, (GC)(4), in the presence of the reducing agent dithiothreitol, the oxidizing ag ent hydrogen peroxide, or the alkylating agent formaldehyde gives a si milar mixture of DNA-drug adducts. Negative ion electrospray mass spec tra indicate that adduct formation involves coupling of the DNA to the anthracycline via a methylene group and that the major adduct is dupl ex DNA containing two molecules of anthracycline, each bound to a sepa rate strand of the DNA via a methylene group. The source of the methyl ene group is formaldehyde. A molecular structure with each anthracycli ne intercalated at a 5'-CpG-3' site and covalently bound from its 3'-a mino group to a 2-amino group of a 2'-deoxyguanosine nucleotide is pro posed based upon spectral data and a relevant crystal structure. The r eaction of (GC)(4) with the anthracyclines and formaldehyde forms an e quilibrium mixture with DNA-drug adducts which is shifted toward free DNA by dilution. The results suggest a pathway to the inhibition of tr anscription by reductively activated adriamycin and daunomycin. Reduct ive activation in the presence of oxygen yields hydrogen peroxide; hyd rogen peroxide oxidizes constituents in the reaction mixture to formal dehyde; and formaldehyde couples the drug to DNA. In this regard, hydr ogen peroxide reacts with adriamycin via Baeyer-Villiger reactions at the 13-position to yield 2, 3, and formaldehyde. Formaldehyde also res ults from hydrogen peroxide oxidation of Tris [tris(hydroxymethyl)amin omethane] present in transcription buffer and spermine, a polyamine co mmonly associated with DNA in vivo, presumably via the Fenton reaction .