Distinct mechanisms of site-specific oxidative DNA damage by doxorubicin in the presence of copper(II) and NADPH-cytochrome P450 reductase.
The anticancer mechanism of doxorubicin (DOX), an anthracycline antibiotic, is believed to involve DNA damage through topoisomerase II inhibition and free radical generation. The free radical generation may also participate in genotoxicity, as well as cardiotoxicity, in normal human cells. The present study showed that DOX generates 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), an indicator of oxidative DNA damage, in HL-60 cells, but not in H(2)O(2)-resistant HP100 cells, suggesting the involvement of H(2)O(2) in cellular DNA damage. Since DOX has both p-quinone and p-hydroquinone residues, free radical generation can be initiated by either reduction or oxidation of DOX. To clarify whether the oxidized or reduced form is more important for DOX-induced H(2)O(2) generation, we investigated the site-specific DNA damage induced by DOX in the presence of Cu(II), in comparison with that in the presence of cytochrome P450 reductase, using (32)P-labeled DNA fragments. DOX caused DNA damage in the presence of Cu(II) or cytochrome P450 reductase. The degree of Cu(II)-mediated DNA damage, including 8-oxodG formation, was much greater than that of cytochrome P450 reductase-mediated DNA damage. DOX plus Cu(II) caused DNA damage specifically at guanine, thymine and cytosine residues, particularly at 5'-GG-3', 5'-GT-3' and 5'-TG-3' sequences. Scavenger experiments suggested the involvement of reactive species generated from H(2)O(2) and Cu(I). When cytochrome P450 reductase and NADPH were used instead of Cu(II), every nucleotide was uniformly damaged, suggesting the participation of.OH. We conclude that DOX may induce carcinostatic and genotoxic effects through oxidation of its p-hydroquinone moiety by metal ion rather than through p-quinone reduction by cytochrome P450 reductase.
Mizutani
Hideki
H
Department of Environmental and Molecular Medicine, Mie University School of Medicine, Edobashi, Tsu, Mie 514-8507, Japan.
Oikawa
Shinji
S
Hiraku
Yusuke
Y
Murata
Mariko
M
Kojima
Michio
M
Kawanishi
Shosuke
S
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000818:Animals; D002417:Cattle; D003300:Copper / Q000494:pharmacology*; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics*; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000032:analysis; D004317:Doxorubicin / Q000633:toxicity*; D016158:Genes, p53 / Q000235:genetics; D018922:HL-60 Cells; D006801:Humans; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D003300:Copper; D004317:Doxorubicin; D000080242:8-Hydroxy-2'-Deoxyguanosine; D009251:NADPH-Ferrihemoprotein Reductase; D003849:Deoxyguanosine