Quantification of formaldehyde-mediated covalent adducts of adriamycin with DNA

Duplex DNA incubated with adriamycin, dithiothreitol (DTT), and Fe3+ under aerobic, aqueous conditions yields double-stranded (DS) DNA bands by denatu ring polyacrylamide gel electrophoresis (DPAGE) analysis, characteristic of DNAs which are interstrand cross-linked. Another laboratory has provided e vidence that formaldehyde produced under these conditions promotes the cova lent linkage of adriamycin to one strand of DNA and suggested that this com plex results in the anomalous DPAGE behavior. We provide herein strong supp ort for this interpretation. We show: (a) that mixtures of DNA and adriamyc in incubated with DTT/Fe3+, H2O2, or formaldehyde all show DS DNA bands on DPAGE, (b) that the DS DNA bands and the formaldehyde-mediated lesion (dete cted by an indirect, GC-MS analysis) form with similar time courses, and in similar amounts, and (c) that the DNA in the DS DNA bands contains approxi mately one such lesion per DNA, whereas the single-stranded DNA is devoid o f it. These results further support the interpretation that adriamycin does not create interstrand cross-links in DNA, and that the DS DNA observed in DPAGE experiments derives from the formaldehyde-mediated monoadduct.