Singlet oxygen ( 1 O 2 ) is a biologically relevant reactive oxygen species capable of efficiently reacting with cellular constituents. The resulting oxidatively generated damage to nucleic acids, membrane unsaturated lipids, and protein components has been shown to be implicated in several diseases, including arthritis, cataracts, and skin cancer. Singlet oxygen may be endogenously produced, among various possibilities, by myeloperoxidase, an enzyme implicated in inflammation processes, and also efficiently in skin by the UVA component of solar radiation through photosensitization reactions. Emphasis is placed in this Review on the description of the main oxidation reactions initiated by 1 O 2 and the resulting modifications within key cellular targets, including guanine for nucleic acids, unsaturated lipids, and targeted amino acids. Most of these reactions give rise to peroxides and dioxetanes, whose formation has been rationalized in terms of [4+2] cycloaddition and 1,2-cycloaddition with dienes + olefins, respectively. The use of [ 18 O]-labeled thermolabile endoperoxides as a source of [ 18 O]-labeled 1 O 2 has been applied to study mechanistic aspects and preferential targets of 1 O 2 in biological systems. A relevant major topic deals with the search for the molecular signature of the 1 O 2 formation in targeted biomolecules within cells. It may be anticipated that [ 18 O]-labeled 1 O 2 and labeled peroxides in association with sensitive mass spectrometric methods should constitute powerful tools for this purpose.

N1-acetyl-N2-formyl-5-methoxykynuramine (AMFK) is a major metabolite of melatonin in mammals. To investigate whether AFMK exists in plants, an aquatic plant, water hyacinth, was used. To achieve this, LC/MS/MS with a deuterated standard was employed. AFMK was identified in any plant for the first time. Both it and its precursor, melatonin, were rhythmic with peaks during the late light phase. These novel rhythms indicate that these molecules do not serve as the chemical signal of darkness as in animals but may relate to processes of photosynthesis or photoprotection. These possibilities are supported by higher production of melatonin and AFMK in plants grown in sunlight (10,000-15,000 microW/cm2) compared to those grown under artificial light (400-450 microW/cm2). Melatonin and AFMK, as potent free radical scavengers, may assist plants in coping with harsh environmental insults, including soil and water pollutants. High levels of melatonin and AFMK in water hyacinth may explain why this plant more easily tolerates environmental pollutants, including toxic chemicals and heavy metals and is successfully used in phytoremediation. These novel findings could lead to improvements in the phytoremediative capacity of plants by either stimulating endogenous melatonin synthesis or by adding melatonin to water/soil in which they are grown.

The decomposition of lipid hydroperoxides into peroxyl radicals is a potential source of singlet oxygen ((1)O(2)) in biological systems. We report herein on evidence of the generation of (1)O(2) from lipid hydroperoxides involving a cyclic mechanism from a linear tetraoxide intermediate proposed by Russell. Using (18)O-labeled linoleic acid hydroperoxide (LA(18)O(18)OH) in the presence of Ce(4+) or Fe(2+), we observed the formation of (18)O-labeled (1)O(2) ((18)[(1)O(2)]) by chemical trapping of (1)O(2) with 9,10-diphenylanthracene (DPA) and detected the corresponding (18)O-labeled DPA endoperoxide (DPA(18)O(18)O) by high-performance liquid chromatography coupled to tandem mass spectrometry. Spectroscopic evidence for the generation of (1)O(2) was obtained by measuring (i) the dimol light emission in the red spectral region (lambda > 570 nm); (ii) the monomol light emission in the near-infrared (IR) region (lambda = 1270 nm); and (iii) the quenching effect of sodium azide. Moreover, the presence of (1)O(2) was unequivocally demonstrated by the direct spectral characterization of the near-IR light emission. For the sake of comparison, (1)O(2) deriving from the H(2)O(2)/OCl(-) and H(2)O(2)/MoO(4)(2)(-) systems or from the thermolysis of the endoperoxide of 1,4-dimethylnaphthalene was also monitored. These chemical trapping and photoemission properties clearly demonstrate that the decomposition of LA(18)O(18)OH generates (18)[(1)O(2)], consistent with the Russell mechanism and pointing to the involvement of (1)O(2) in lipid hydroperoxide mediated cytotoxicity.

The aim of the present work was to evaluate the potential for 1 O 2 to induce oxidation of cellular DNA. For this purpose cells were incubated in the presence of a water-soluble endoperoxide whose thermal decomposition leads to the formation of singlet oxygen. Thereafter, DNA was extracted and the level of several modified DNA bases was determined by HPLC analysis coupled to a tandem mass spectrometric detection. A significant increase in the level of 8-oxo-7,8-dihydro-2-deoxyguanosine was observed upon incubation of the cells with the chemical generator of Modification of cellular DNA upon exposure to reactive oxygen and nitrogen species is the likely initial event involved in the induction of the mutagenic and lethal effects of various oxidative stress agents (1-3). As an example, the deleterious effects of UVA radiation are, at least partly, explained in terms of photooxidation of cellular DNA (4, 5). The mechanism of UVA-mediated photooxidative damage to DNA is not completely established. Evidence has been accumulated over the years for the significant implication of singlet oxygen, as the result of UVA activation (4, 6) of endogenous photosensitizers (porphyrins, flavins, . . . ) not yet characterized. However, a type I mechanism involving the initial formation of a DNA radical cation, that could be predominantly located at guanine sites due the lowest ionization potential of the latter base and/or to the possibility of charge transfer in DNA (7), could not be excluded. To our knowledge, no clear evidence has been provided to demonstrate that singlet oxygen is able to oxidize cellular DNA. It should be added, however, that 1 O 2 is known to be mutagenic and genotoxic (2,3,8). In addition, singlet oxygen has been identified as the reactive oxygen species involved in numerous biological processes. Among others we may cite neutrophils phagocytosis (9) and enzymatic processes (10).Reactions of singlet oxygen with nucleosides and isolated DNA are well documented. Interestingly, it was shown that 1 O 2 oxidizes, among the nucleosides, almost exclusively the guanine base. Singlet oxygen reacts with free dGuo 1 and short oligonucleotides to give rise to the overwhelming formation of the 4R and 4S diastereomers of 4-hydroxy-8-oxo-4,8-dihydro-2Ј-deoxyguanosine, together with a small amount of 8-oxodGuo (11-15). In contrast, 8-oxodGuo was found to be the major oxidation product formed upon exposure of isolated DNA to 1 O 2 (6, 11, 16). However, 8-oxodGuo cannot be considered as a specific biological marker of 1 O 2 , since this DNA lesion could be formed under various conditions of oxidative stress, including those generated by one-electron process (17), hydroxyl radical (18), and Fenton-type reactions (19). On one hand, this explains why 8-oxodGuo could be used as an ubiquitous biomarker of DNA oxidation (20 -22). On the other hand, the formation of 8-oxodGuo in cellular DNA could not be attributed to the initial formation of 1 O 2 . During the recent past, water-soluble generators of 1 O 2 , that consist of aromatic...

This survey focuses on recent aspects of the singlet oxygen oxidation of the guanine moiety of nucleosides, oligonucleotides, isolated and cellular DNA that has been shown to be the exclusive DNA target for this biologically relevant photogenerated oxidant. A large body of mechanistic data is now available from studies performed on nucleosides in both aprotic solvents and aqueous solutions. A common process to both reaction conditions is the formation of 8-oxo-7,8-dihydroguanine by reduction of 8-hydroperoxyguanine that arises from the rearrangement of initially formed endoperoxide across the 4,8-bond of the purine moiety. However, in organic solvent the hydroperoxide is converted as a major degradation pathway into a dioxirane that subsequently decomposes into a complex pattern of oxidation products. A different reaction that involved the formation of a highly reactive quinonoid intermediate consecutively to the loss of a water molecule from the 8-hydroperoxide has been shown to occur in aqueous solution. Subsequent addition of a water molecule at C5 leads to the generation of a spiroiminodihy-dantoin compound via a rearrangement that involves an acyl shift. However, in both isolated and cellular DNA the latter decomposition pathway is at the best a minor process, because only 8-oxo-7,8-dihydroguanine has been found to be generated. It is interesting to point out that singlet oxygen has been shown to contribute predominantly to the formation of 8-oxo-7,8-dihydroguanine in the DNA of bacterial and human cells upon exposure to UVA radiation. It may be added that the formation of secondary singlet-oxygen oxidation products of 8-oxo-7,8-dihydroguanine, including spiroiminodihydantoin and oxaluric acid that were characterized in nucleosides and oligonucleotide, respectively, have not yet been found in cellular DNA.

Oxidative reactions of DNA commonly result in base modifications. Among the four DNA bases, guanine is the most susceptible to oxidation, and one of its main oxidized compounds, namely 8-oxo-7,8-dihydroguanine (8-oxoGua), has been extensively studied in terms of formation, repair, and mutagenicity. However, the latter modified purine base is readily subjected to further oxidation reactions which have recently become a matter of interest. Emphasis was placed in this work on the identification of the final singlet oxygen oxidation products of 8-oxoGua in single-stranded DNA. Oxaluric acid was found to be the predominant product of the reaction. Insights in the mechanistic pattern of oxaluric acid formation were gained from isotopic labeling experiments in association with mass spectrometry measurements. It was found that oxaluric acid is formed via an oxidized guanidinohydantoin intermediate, arising from the likely degradation of a transient 5-hydroperoxide. Two subsequent hydrolytic steps that are accompanied by the release of guanidine are likely to be involved in the formation of oxaluric acid.

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The reaction of hypochlorous acid (HOCl) with hydrogen peroxide is known to generate stoichiometric amounts of singlet molecular oxygen [O2 ( 1 ⌬g)]. This study shows that HOCl can also react with linoleic acid hydroperoxide (LAOOH), generating O2 ( 1 ⌬g) with a yield of 13 ؎ 2% at physiological pH. Characteristic light emission at 1,270 nm, corresponding to O2 ( 1 ⌬g) monomolecular decay, was observed when HOCl was reacted with LAOOH or with liposomes containing phosphatidylcholine hydroperoxides, but not with cumene hydroperoxide or tert-butyl hydroperoxide. The generation of O2 ( 1 ⌬g lipid hydroperoxides ͉ mass spectrometry ͉ myeloperoxidase ͉ near-infrared emission ͉ 18 O-labeled oxygen