Hydroxyurea induces site-specific DNA damage via formation of hydrogen peroxide and nitric oxide

Hydroxyurea is a chemotherapeutic agent used for the treatment of myeloprol iferative disorders (MPD) and solid tumors. The mutagenic and carcinogenic potential of hydroxyurea has not been established, although hydroxyurea has been associated with an increased risk of leukemia in MPD patients. To cla rify whether hydroxyurea has potential carcinogenicity, we examined site-sp ecific DNA damage induced by dromrurea using P-32-5'-end-labeled DNA fragme nts obtained from the human p53 and p16 tumor suppressor genes and the c-Ha -ras-1 protooncogene. Hydroxyurea caused Cu(II)-mediated DNA damage especia lly at thy mine and cytosine residues. NADH efficiently enhanced hydroxyure a-induced DNA damage. The DNA damage was almost entirely inhibited by catal ase and bathocuproine, a Cu(I)-specific chelator, suggesting the involvemen t of hydrogen peroxide (H2O2) and Cu(I). Typical free hydroxyl radical scav engers did not inhibit DNA damage by hydroxyurea, but methional did. These results suggest that crypto-hydroxyl radicals such as CU(I)-hydroperoxo com plex (Cu(I)-OOH) cause DNA damage. Formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) was induced by hydroxyurea in the presence of Cu(II). An electron spin resonance spectroscopic study using N-(dithiocarboxy)sarcosine as a n itric oxide (NO)-trapping reagent demonstrated that NO was generated from h ydroxyurea in the presence and absence of catalase. In addition. the genera tion of formamide was detected by both gas chromatography-mass spectrometry (GC-MS) and time-of-flight-mass spectrometry (TOF-MS). A high concentratio n of hydroxyurea induced depurination at DNA bases in an H2O2-independent m anner, and endonuclease IV treatment led to chain cleavages. These results suggest that hydroxyurea could induce base oxidation as the major pathway o f DNA modification and depurination as a minor pathway. Therefore, it is co nsidered that DNA damage by hydroxyurea participates in not only anti-cance r activity, but also carcinogenesis.