Hydroxyurea Induces Site‐specific DNA Damage via Formation of Hydrogen Peroxide and Nitric Oxide

Hydroxyurea is a chemotherapeutic agent used for the treatment of myeloproliferative 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 clarify whether hydroxyurea has potential carcinogenicity, we examined site‐specific DNA damage induced by hydroxyurea using (32)P‐5′‐end‐labeled DNA fragments obtained from the human p53 and p16 tumor suppressor genes and the c‐Ha‐ras‐1 protooncogene. Hydroxyurea caused Cu(II)‐mediated DNA damage especially at thymine and cytosine residues. NADH efficiently enhanced hydroxyurea‐induced DNA damage. The DNA damage was almost entirely inhibited by catalase and bathocuproine, a Cu(I)‐specific chelator, suggesting the involvement of hydrogen peroxide (H(2)O(2)) and Cu(I). Typical free hydroxyl radical scavengers did not inhibit DNA damage by hydroxyurea, but methional did. These results suggest that crypto‐hydroxyl radicals such as Cu(I)‐hydroperoxo complex (Cu(I)‐OOH) cause DNA damage. Formation of 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) was induced by hydroxyurea in the presence of Cu(H). An electron spin resonance spectroscopic study using N‐(dithiocarboxy)sarcosine as a nitric oxide (NO)‐trapping reagent demonstrated that NO was generated from hydroxyurea in the presence and absence of catalase. In addition, the generation of formamide was detected by both gas chromatography‐mass spectrometry (GC‐MS) and time‐of‐flight‐mass spectrometry (TOF‐MS). A high concentration of hydroxyurea induced depurination at DNA bases in an H(2)O(2)‐independent manner, and endonu‐clease IV treatment led to chain cleavages. These results suggest that hydroxyurea could induce base oxidation as the major pathway of DNA modification and depurination as a minor pathway. Therefore, it is considered that DNA damage by hydroxyurea participates in not only anti‐cancer activity, but also carcinogenesis.