Reduction of adriamycin to a semiquinone-free radical by NADPH cytochrome P-450 reductase produces DNA cleavage in a reaction mediated by molecular oxygen

Abstract

Adriamycin is reduced to a semiquinone-free radical by microsomal and nuclear enzymes. To investigate the effect of these radicals on DNA, an end-labeled DNA fragment of defined sequence was included in a reaction that contained NADPH, NADPH cytochrome P-450 reductase (EC 1.6.2.4), and adriamycin. Extensive DNA strand scission was observed. The breaks occurred at all nucleotides with equal probability regardless of nucleotide sequence. Single-stranded DNA was as good a substrate as double-stranded DNA for the strand scission reaction. DNA damage occurred during the early phase of the reaction. No strand breaks were observed upon addition of DNA to the reaction 60 min after enzyme activation despite the high concentration of stable free radicals present at this time. The extent of strand scission was greatly reduced when the reaction was carried out in a nitrogen atmosphere. We conclude that DNA breakage created by enzymatically derived adriamycin-free radicals is mediated by molecular oxygen, most probably by hydroxyl-free radicals and hydroxyl ions. We also conclude that the semiquinone-free radicals of adriamycin and radicals of the reduced forms of adriamycin need not intercalate into DNA to create strand breaks.