DNA base modifications induced in isolated human chromatin by NADH dehydrogenase-catalyzed reduction of doxorubicin.
The antineoplastic benzanthroquinone drug doxorubicin can undergo flavoenzyme-catalyzed one-electron reduction which, in an aerobic environment, leads to the generation of oxygen-derived species. We therefore sought to determine whether doxorubicin in the presence of NADH dehydrogenase and the transition metal ions Fe(III) or Cu(II) induces DNA base modifications in isolated human chromatin. NADH dehydrogenase-catalyzed reduction of doxorubicin (25-100 microM) caused hydroxyl radical production detected as methane generated from dimethyl sulfoxide; addition of isolated human chromatin to the system produced a concentration-dependent quenching of detectable hydroxyl radical formation. Doxorubicin (5-50 microM)-stimulated enzyme-catalyzed oxidation of NADH was also diminished, but still detectable, in the presence of chromatin. Doxorubicin-induced DNA base modifications in chromatin were measured by gas chromatography/mass spectrometry with selected-ion monitoring. Production of modified bases required the addition of transition metal ion and was enhanced by the addition of active flavoenzyme. The non-redox cycling analogue 5-iminodaunorubicin induced significantly less base modification than did doxorubicin. In the presence of Fe(III), NADH dehydrogenase-catalyzed reduction of doxorubicin caused enhancement in the content of all modified bases over control levels. Substitution of Cu(II) for Fe(III) altered both the degree and the pattern of doxorubicin/NADH dehydrogenase-induced base modifications. The scavengers of hydroxyl radical mannitol and dimethyl sulfoxide or catalase did not significantly affect doxorubicin/NADH/NADH dehydrogenase/transition metal ion-induced base modifications. Superoxide dismutase further enhanced production of all base modifications. The data demonstrate that flavoenzyme-catalyzed redox cycling of doxorubicin generates typical hydroxyl radical-induced base modifications in the DNA of isolated human chromatin, suggesting a possible mechanism for the mutagenicity of doxorubicin in vivo.
Akman
S A
SA
Department of Medical Oncology and Therapeutics Research, City of Hope National Medical Center, Duarte, California 91010.
Doroshow
J H
JH
Burke
T G
TG
Dizdaroglu
M
M
D002843:Chromatin / Q000737:chemistry* / Q000378:metabolism; D003300:Copper / Q000494:pharmacology; D004247:DNA / Q000737:chemistry* / Q000187:drug effects; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004317:Doxorubicin / Q000378:metabolism / Q000494:pharmacology*; D016166:Free Radical Scavengers; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D006878:Hydroxides / Q000378:metabolism / Q000494:pharmacology; D017665:Hydroxyl Radical; D007501:Iron / Q000494:pharmacology; D008353:Mannitol / Q000494:pharmacology; D009243:NAD / Q000378:metabolism; D009245:NADH Dehydrogenase / Q000378:metabolism*; D010084:Oxidation-Reduction
D002843:Chromatin; D016166:Free Radical Scavengers; D006878:Hydroxides; D009243:NAD; D017665:Hydroxyl Radical; D008353:Mannitol; D003300:Copper; D004317:Doxorubicin; D004247:DNA; D007501:Iron; D009245:NADH Dehydrogenase; D004121:Dimethyl Sulfoxide