M.H.G. Medeiros

Singlet oxygen-mediated damage to cellular DNA determined by the comet assay associated with DNA repair enzymes.

Abstract The damage profile produced by the reaction of singlet molecular oxygen with cellular DNA was determined using the comet assay associated with DNA repair enzymes. Singlet oxygen was produced intracellularly by thermal decomposition of a watersoluble endoperoxide of a naphthalene derivative which is able to penetrate through the membrane into mammalian cells. We found that the DNA modifications produced by singlet oxygen were almost exclusively oxidised purines recognised by the formamidopyrimidine DNA N-glycosylase. In contrast, significant amounts of direct strand breaks and alkalilabile sites or oxidised pyrimidines, detectable by the bacterial endonuclease III, were not produced.

[18O]-labeled singlet oxygen as a tool for mechanistic studies of 8-oxo-7,8-dihydroguanine oxidative damage: detection of spiroiminodihydantoin, imidazolone and oxazolone derivatives.

Abstract A watersoluble [18O]labeled endoperoxide derived from N,Ndi(2,3-dihydroxypropyl)-1,4-naphthalenedipropanamide (DHPN18O2) has been shown to act as a clean chemical source of [18O]labeled molecular singlet oxygen. This allows the assessment of the singlet oxygen (1O2) reactivity toward biological targets such as DNA. The present work focuses on the qualitative identification of the main 1O2-oxidation products of 8-oxo-7,8-dihydro-2deoxyguanosine, which was achieved using high performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLCESIMS/MS). Thus, the [18O]labeled and unlabeled imidazolone and oxazolone, together with the diastereoisomeric spiroiminodihydantoin nucleosides, were detected as the main degradation products. In addition, a modified nucleoside that exhibits similar features as those of the oxidized guanidinohydantoin molecule was detected. Our data strongly suggest that the imidazolone and oxazolone nucleosides are generated via the rearrangement of an unstable 5-hydroperoxide intermediate. Interestingly, the combined use of appropriate tools, including isotopically labeled singlet oxygen and the high resolution HPLCESIMS/MS technique, has allowed to shed new light on the 1O2- mediated oxidation reactions of guanine DNA components.