Thymine hydroperoxide as a potential source of singlet molecular oxygen in DNA

The decomposition of organic hydroperoxides into peroxyl radicals is a potential source of singlet molecular oxygen [O2 (1Deltag)] in biological systems. This study shows that 5-(hydroperoxymethyl)uracil (5-HPMU), a thymine hydroperoxide within DNA, reacts with metal ions or HOCl, generating O2 (1Deltag). Spectroscopic evidence for generation of O2 (1Deltag) was obtained by measuring (i) the bimolecular decay, (ii) the monomolecular decay, and (iii) the observation of D2O enhancement of O2 (1Deltag) production and the quenching effect of NaN3. Moreover, the presence of O2 (1Deltag) was unequivocally demonstrated by the direct characterization of the near-infrared light emission. For the sake of comparison, O2 (1Deltag) derived from the H2O2/HOCl system and from the thermolysis of the N,N'-di(2,3-dihydroxypropyl)-1,4-naphthalenedipropanamide endoperoxide was also monitored. More evidence of O2 (1Deltag) generation was obtained by chemical trapping of O2 (1Deltag) with anthracene-9,10-divinylsulfonate (AVS) and detection of the specific AVS endoperoxide by HPLC/MS/MS. The detection by HPLC/MS of 5-(hydroxymethyl)uracil and 5-formyluracil, two thymine oxidation products generated from the reaction of 5-HPMU and Ce4+ ions, supports the Russell mechanism. These photoemission properties and chemical trapping clearly demonstrate that the decomposition of 5-HPMU generates O2 (1Deltag) by the Russell mechanism and point to the involvement of O2 (1Deltag) in thymidine hydroperoxide cytotoxicity.