NITRIC-OXIDE GENERATION FROM HYDROXYUREA VIA COPPER-CATALYZED PEROXIDATION AND IMPLICATIONS FOR PHARMACOLOGICAL ACTIONS OF HYDROXYUREA

We investigated the generation of nitric oxide ((NO)-N-.) by H2O2-depe ndent peroxidation of hydroxyurea in the presence of copper-containing proteins such as Cu,Zn-superoxide dismutase (Cu,Zn-SOD) or ceruloplas min as a catalyst. In the reaction mixture of hydroxyurea, Cu,Zn-SOD, and H2O2, (NO)-N-. generation was identified by measuring the specific electron spin resonance (ESR) signal of 2-phenyl-4,4,5,5-tetramethyli midazoline-1-oxyl 3-oxide (PTIO). The ESR signal of the NO-hemoglobin adduct was also detected in human red blood cells during copper-cataly zed peroxidation of hydroxyurea. The (NO)-N-. production during peroxi dation of hydroxyurea was quantified as NO2- formation, measured by us ing the Griess assay, and the amount of NO2- was dependent on the conc entration of hydroxyurea of the reaction mixture. ESR spin trapping wi th 5,5-dimethyl-1-pyrroline N-oxide (DMPO) showed hydroxy radical ((OH )-O-.) generation in the reaction of H2O2 With either Cu,Zn-SOD or cer uloplasmin. Several (OH)-O-. scavengers, such as ethanol, thiourea, DM PO, and dimethylsulfoxide, and the metal-chelating agent diethylenetri aminepentaacetic acid significantly inhibited (NO)-N-. generation from hydroxyurea. This indicates that (NO)-N-. release from hydroxyurea ma y be mediated by (OH)-O-. derived from the copper-catalyzed Fenton-lik e reaction. Incubation of hydroxyurea and Cu,Zn-SOD with xanthine oxid ase and hypoxanthine in a system forming O-2(-) --> H2O2 also resulted in appreciable (NO)-N-. production. These results suggest that (NO)-N -. production from hydroxyurea catalyzed by copper-containing proteins may be the molecular basis of the pharmacological and antitumor actio n of hydroxyurea.