Glutathione modifies the oxidation products of 2'-deoxyguanosine by singlet molecular oxygen.

The oxidation of the free nucleoside 2'-deoxyguanosine (dGuo) by singlet molecular oxygen ((1)O2) has been studied over the three last decades due to the major role of DNA oxidation products in process such as ageing, mutation and carcinogenesis. In the present work we investigated the dGuo oxidation by (1)O2 in the presence of the important low molecular antioxidant, glutathione, in its reduced (GSH) and oxidized (GSSG) forms. There were applied different conditions of concentration, pH, time of incubation, and the use of a [(18)O]-labeled thermolabile endoperoxide naphthalene derivative as a source of [(18)O]-labeled (1)O2. Data was obtained through high performance liquid chromatography (HPLC) and HPLC coupled to micrOTOF Q-II analysis of the main oxidation products: the diastereomers of spiroiminodihydantoin-2'-deoxyribonucleosides (dSp) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo). An intriguing result was that 8-oxodGuo levels increased by 100 fold when dGuo was oxidized by (1)O2 in the presence of GSH and by 2 fold in the presence of GSSG, while dSp levels dropped to zero for both conditions. All data from dGuo, 8-oxodGuo and dSp quantification together with the analysis of residual GSH/GSSG content in each sample strongly suggest that glutathione modifies the mechanism of dGuo oxidation by (1)O2 by disfavoring the pathway of dSp formation.

Generation of Singlet Molecular Oxygen by Lipid Hydroperoxides and Nitronium Ion†.

Singlet molecular oxygen is a reactive species involved in biological oxidative processes. The major cellular targets of singlet molecular oxygen are unsaturated fatty acids in the membrane, as well as nucleic acids and proteins. The aim of this study was to investigate whether lipids and commercial hydroperoxides generate singlet molecular oxygen, in presence of nitronium and activated nitronium ion. For this purpose, monomol light emitted in the near-infrared region (λ = 1270 nm) was used to monitor singlet molecular oxygen decay in different solvents, with different hydroperoxides and in the presence of azide. Direct measurements of the singlet molecular oxygen spectrum at 1270 nm recorded during the reaction between lipids and commercial hydroperoxides and nitronium ions unequivocally demonstrated the formation of this excited species.

Singlet molecular oxygen generation by light-activated DHN-melanin of the fungal pathogen Mycosphaerella fijiensis in black Sigatoka disease of bananas.

In pathogenic fungi, melanin contributes to virulence, allowing tissue invasion and inactivation of the plant defence system, but has never been implicated as a factor for host cell death, or as a light-activated phytotoxin. Our research shows that melanin synthesized by the fungal banana pathogen Mycosphaerella fijiensis acts as a virulence factor through the photogeneration of singlet molecular oxygen O2 (1Δg). Using analytical tools, including elemental analysis, ultraviolet/infrared absorption spectrophometry and MALDI-TOF mass spectrometry analysis, we characterized both pigment content in mycelia and secreted to the culture media as 1,8-dihydroxynaphthalene (DHN)-melanin type compound. This is sole melanin-type in M. fijiensis. Isolated melanins irradiated with a Nd:YAG laser at 532 nm produced monomol light emission at 1270 nm, confirming generation of O2 (1Δg), a highly reactive oxygen specie (ROS) that causes cellular death by reacting with all cellular macromolecules. Intermediary polyketides accumulated in culture media by using tricyclazole and pyroquilon (two inhibitors of DHN-melanin synthesis) were identified by ESI-HPLC-MS/MS. Additionally, irradiation at 532 nm of that mixture of compounds and whole melanized mycelium also generated O2 (1Δg). A pigmented-strain generated more O2 (1Δg) than a strain with low melanin content. Banana leaves of cultivar Cavendish, naturally infected with different stages of black Sigatoka disease, were collected from field. Direct staining of the naturally infected leaf tissues showed the presence of melanin that was positively correlated to the disease stage. We also found hydrogen peroxide (H2O2) but we cannot distinguish the source. Our results suggest that O2 (1Δg) photogenerated by DHN-melanin may be involved in the destructive effects of Mycosphaerella fijiensis on banana leaf tissues. Further studies are needed to fully evaluate contributions of melanin-mediated ROS to microbial pathogenesis.