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

Abstract

The decomposition of organic hydroperoxides into peroxyl radicals is a potential source of singlet molecular oxygen [O 2 ( 1Δ g)] in biological systems. This study shows that 5-(hydroperoxymethyl)uracil (5-HPMU), a thymine hydroperoxide within DNA, reacts with metal ions or HOCl, generating O 2 ( 1Δ g). Spectroscopic evidence for generation of O 2 ( 1Δ g) was obtained by measuring (i) the bimolecular decay, (ii) the monomolecular decay, and (iii) the observation of D 2O enhancement of O 2 ( 1Δ g) production and the quenching effect of NaN 3. Moreover, the presence of O 2 ( 1Δ g) was unequivocally demonstrated by the direct characterization of the near-infrared light emission. For the sake of comparison, O 2 ( 1Δ g) derived from the H 2O 2/HOCl system and from the thermolysis of the N,N′-di(2,3-dihydroxypropyl)-1,4-naphthalenedipropanamide endoperoxide was also monitored. More evidence of O 2 ( 1Δ g) generation was obtained by chemical trapping of O 2 ( 1Δ g) 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 Ce 4+ ions, supports the Russell mechanism. These photoemission properties and chemical trapping clearly demonstrate that the decomposition of 5-HPMU generates O 2 ( 1Δ g) by the Russell mechanism and point to the involvement of O 2 ( 1Δ g) in thymidine hydroperoxide cytotoxicity.