Paolo Di Mascio

Lycopene as the most efficient biological carotenoid singlet oxygen quencher

Lycopene, a biologically occurring carotenoid, exhibits the highest physical quenching rate constant with singlet oxygen (kq = 31 X 10(9) M-1 s-1), and its plasma level is slightly higher than that of beta-carotene (kq = 14 X 10(9) M-1 s-1). This is of considerable general interest, since nutritional carotenoids, particularly beta-carotene, and other antioxidants such as alpha-tocopherol (kq = 0.3 X 10(9) M-1 s-1) have been implicated in the defense against prooxidant states; epidemiological evidence reveals that such compounds exert a protective action against certain types of cancer. Also, albumin-bound bilirubin is a known singlet oxygen quencher (kq = 3.2 X 10(9) M-1 s-1). Interestingly, those compounds with low kq values occur at higher plasma levels. When these differences are taken into account, the singlet oxygen quenching capacities of lycopene (0.7 microM in plasma), beta-carotene (0.5 microM in plasma), albumin-bound bilirubin (15 microM in plasma), and alpha-tocopherol (22 microM in plasma) are of comparable magnitude.

Novel rhythms of N1-acetyl-N2-formyl-5-methoxykynuramine and its precursor melatonin in water hyacinth: importance for phytoremediation

N1‐acetyl‐N2‐formyl‐5‐methoxykynura‐mine (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 pho‐toprotection. These possibilities are supported by higher production of melatonin and AFMK in plants grown in sunlight (10, 000–15, 000 μW/cm2) compared to those grown under artificial light (400–450 μW/ 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.—Dun‐Xian Tan, Lucien C. Manchester, Paolo Di Mascio, Glaucia R. Martinez, Fernanda M. Prado, and Russel J. Reiter. Novel rhythms of N1‐acetyl‐N2‐formyl‐5‐methoxykynuramine and its precursor mel‐atonin in water hyacinth: importance for phytoremedia‐tion. FASEB J. 21, 1724–1729 (2007)

SINGLET MOLECULAR OXYGEN PRODUCTION IN THE REACTION OF PEROXYNITRITE WITH HYDROGEN PEROXIDE

Peroxynitrite and hydrogen peroxide are mediators of cytotoxicity. This study shows that the peroxynitrite anion reacts with hydrogen peroxide to release oxygen accompanied by emission of chemiluminescence (CL). Direct characterization of this light emission attributes it to the transition of singlet molecular oxygen to the triplet ground state. Chemiluminescence was monitored: (i) by dimol light emission in the red spectral region (> 610 nm) using a red‐sensitive photomultiplier; and (ii) by monomol light emission in the infrared (1270 nm) with a liquid nitrogen‐cooled germanium diode. These properties of photoemission and the enhancing effect of deuterium oxide on CL intensity as well as the quenching effect of sodium azide are diagnostic of molecular oxygen in the excited singlet state. For comparison, singlet molecular oxygen arising from the thermolysis of the water‐soluble endoperoxide of 3,3′‐(1,4‐naphthylidene)dipropionate or from the hypochlorite/H2O2 system was also monitored. These novel observations identify a potential singlet oxygen‐dependent mechanism contributing to cytotoxicity mediated by peroxynitrite and hydrogen peroxide.

Singlet Oxygen Oxidation of Isolated and Cellular DNA: Product Formation and Mechanistic Insights

Abstract 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 spiroiminodihydantoin 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.

Biological hydroperoxides and singlet molecular oxygen generation

The decomposition of lipid hydroperoxides (LOOH) into peroxyl radicals is a potential source of singlet molecular oxygen (1O2) in biological systems. Recently, we have clearly demonstrated the generation of 1O2 in the reaction of lipid hydroperoxides with biologically important oxidants such as metal ions, peroxynitrite and hypochlorous acid. The approach used to unequivocally demonstrate the generation of 1O2 in these reactions was the use of an isotopic labeled hydroperoxide, the 18O‐labeled linoleic acid hydroperoxide, the detection of labeled compounds by HPLC coupled to tandem mass spectrometry (HPLC‐MS/MS) and the direct spectroscopic detection and characterization of 1O2 light emission. Using this approach we have observed the formation of 18O‐labeled 1O2 by chemical trapping of 1O2 with anthracene derivatives and detection of the corresponding labeled endoperoxide by HPLC‐MS/MS. The generation of 1O2 was also demonstrated by direct spectral characterization of 1O2 monomol light emission in the near‐infrared region (λ = 1270 nm). In summary, our studies demonstrated that LOOH can originate 1O2. The experimental evidences indicate that 1O2 is generated at a yield close to 10% by the Russell mechanism, where a linear tetraoxide intermediate is formed in the combination of two peroxyl radicals. In addition to LOOH, other biological hydroperoxides, including hydroperoxides formed in proteins and nucleic acids, may also participate in reactions leading to the generation 1O2. This hypothesis is currently being investigated in our laboratory.

Tryptophan Oxidation by Singlet Molecular Oxygen [O2 (1Δg)]: Mechanistic Studies Using 18O-Labeled Hydroperoxides, Mass Spectrometry, and Light Emission Measurements

Proteins have been considered important targets for reactive oxygen species. Indeed, tryptophan (W) has been shown to be a highly susceptible amino acid to many oxidizing agents, including singlet molecular oxygen [O2(1Deltag)]. In this study, two cis- and trans-tryptophan hydroperoxide (WOOH) isomers were completely characterized by HPLC/mass spectrometry and NMR analyses as the major W-oxidation photoproducts. These photoproducts underwent thermal decay into the corresponding alcohols. Additionally, WOOHs were shown to decompose under heating or basification, leading to the formation of N-formylkynurenine (FMK). Using 18O-labeled hydroperoxides (W18O18OH), it was possible to confirm the formation of two oxygen-labeled FMK molecules derived from W18O18OH decomposition. This result demonstrates that both oxygen atoms in FMK are derived from the hydroperoxide group. In addition, these reactions are chemiluminescent (CL), indicating a dioxetane cleavage pathway. This mechanism was confirmed since the CL spectrum of the WOOH decomposition matched the FMK fluorescence spectrum, unequivocally identifying FMK as the emitting species.

Inhibition of 5-aminolevulinic acid-induced DNA damage by melatonin, N1-acetyl-N2-formyl-5-methoxykynuramine, quercetin or resveratrol

Abstract:  Porphyrias are defined as either inborn or acquired diseases related to enzymatic deficiencies in the heme biosynthetic pathway. Lead poisoning, hereditary tyrosinemia, and acute intermittent porphyria (AIP) are characterized by the absence of photosensitivity and the accumulation of 5‐aminolevulinic acid (ALA) together with its increased urinary excretion. The main clinical manifestations of AIP are intermittent attacks of abdominal pain, neuromuscular weaknesses and neuropsychiatry alterations, and also an association with primary liver cancer, in which may be involved the oxidative potential of ALA which is able to cause DNA damage. The use of antioxidants in the treatment of ALA‐induced oxidative stress is not well established. In the current work, we show the antioxidant efficacy of several compounds including melatonin, quercetin, resveratrol and N1‐acetyl‐N2‐formyl‐5‐methoxykynuramine (AFMK), a melatonin oxidation product, in terms of their ability to limit DNA damage induced by ALA/Fe2+ in an in vitro system. Damage was measured by plasmid DNA strand breaks and detection of 8‐oxo, 7‐8‐dihydro,2′‐deoxyguanosine (8‐oxodGuo) by high‐performance liquid chromatography coupled with electrochemical detection. All compounds tested showed a dose‐dependent protective action against free radical damage. These results could be the first step toward studies of the possible use of these antioxidants in oxidative stress promoted by ALA or other pro‐oxidants.

Hydroxyl radicals are involved in the oxidation of isolated and cellular DNA bases by 5-aminolevulinic acid.

5‐Aminolevulinic acid (ALA) is a heme precursor, pathological accumulation of which is associated with liver cancer. We show that the reactive oxygen species produced upon ALA metal‐catalyzed oxidation promote the formation of several radical‐induced base degradation products in isolated DNA. The distribution of modified bases is similar to that obtained upon gamma irradiation. This observation strongly suggests the involvement of hydroxyl radicals in the ALA‐mediated DNA damage. Increased levels of 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine and 5‐hydroxy‐2′‐deoxycytidine in organ DNA of rats chronically treated with ALA were observed. This is strongly suggestive of the implication of hydroxyl radicals in the ALA‐induced degradation of cellular DNA.

Damage to isolated DNA mediated by singlet oxygen

In the present work, we study the reaction of singlet oxygen (1O2) with isolated DNA. Emphasis is placed on the identification and quantitative measurement of the DNA modifications that are produced by the reaction of 1O2 with DNA. For this purpose, calf-thymus DNA was incubated with the endoperoxide of N,N′-di(2,3-dihydroxypropyl)-1,4-naphthalenedipropanamide, a chemical generator of 1O2. Thereafter, DNA was digested, and the resulting oxidized nucleosides were measured by means of a recently optimized high-performance-liquid-chromatography tandem-mass-spectrometry assay. It was found that, among the different DNA lesions observed, 7,8-dihydro-8-oxo-2′-deoxyguanosine is the major 1O2-mediated DNA-damage product. Interestingly, cyclobutane pyrimidine dimers, oxidized pyrimidine bases, 7,8-dihydro-8-oxo-2′-deoxyadenosine, and 2,6-diamino-5-formamido-4-hydroxypyrimidine are not formed, at least not in detectable amounts, following treatment of DNA with the 1O2 generator. The reported results strongly suggest that the decomposition of the endoperoxide provides a pure source of 1O2, and that reaction of 1O2 with isolated DNA induces the specific formation of 7,8-dihydro-8-oxo-2′-deoxyguanosine.

DNA damage by singlet oxygen and cellular protective mechanisms

Reactive oxygen species, as singlet oxygen ((1)O(2)) and hydrogen peroxide, are continuously generated by aerobic organisms, and react actively with biomolecules. At excessive amounts, (1)O(2) induces oxidative stress and shows carcinogenic and toxic effects due to oxidation of lipids, proteins and nucleic acids. Singlet oxygen is able to react with DNA molecule and may induce G to T transversions due to 8-oxodG generation. The nucleotide excision repair, base excision repair and mismatch repair have been implicated in the correction of DNA lesions induced by (1)O(2) both in prokaryotic and in eukaryotic cells. (1)O(2) is also able to induce the expression of genes involved with the cellular responses to oxidative stress, such as NF-κB, c-fos and c-jun, and genes involved with tissue damage and inflammation, as ICAM-1, interleukins 1 and 6. The studies outlined in this review reinforce the idea that (1)O(2) is one of the more dangerous reactive oxygen species to the cells, and deserves our attention.