Search

In mammalian tissues, ultraweak chemiluminescence arising from biomolecule oxidation has been attributed to the radiative deactivation of singlet molecular oxygen [O2 (1Δg)] and electronically excited triplet carbonyl products involving dioxetane intermediates. Herein, we describe evidence of the generation of O2 (1Δg) in aqueous solution via energy transfer from excited triplet acetone. This involves thermolysis of 3,3,4,4-tetramethyl-1,2-dioxetane, a chemical source, and horseradish peroxidase-catalyzed oxidation of 2-methylpropanal, as an enzymatic source. Both sources of excited carbonyls showed characteristic light emission at 1,270 nm, directly indicative of the monomolecular decay of O2 (1Δg). Indirect analysis of O2 (1Δg) by electron paramagnetic resonance using the chemical trap 2,2,6,6-tetramethylpiperidine showed the formation of 2,2,6,6-tetramethylpiperidine-1-oxyl. Using [18O]-labeled triplet, ground state molecular oxygen [18O2 (3∑g -)], chemical trapping of 18O2 (1Δg) with disodium salt of anthracene-9,10-diyldiethane-2,1-diyl disulfate yielding the corresponding double-[18O]-labeled 9,10-endoperoxide, was detected through mass spectrometry. This corroborates formation of O2 (1Δg). Altogether, photoemission and chemical trapping studies clearly demonstrate that chemically and enzymatically nascent excited carbonyl generates 18O2 (1Δg) by triplet-2 (3∑g -), and supports the long formulated hypothesis of O2 (1Δg) involvement in physiological and pathophysiological events that might take place in tissues in the absence of light. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cholesterol (Ch) can be oxidized by reactive oxygen species, forming oxidized products such as Ch hydroperoxides (ChOOH). These hydroperoxides can disseminate the peroxidative stress to other cell compartments. In this work, the ability of ChOOH to induce strand breaks and/or base modifications in a plasmid DNA model was evaluated. In addition, HPLC/MS/MS analyses were performed to investigate the formation of 8-oxo-7,8-dihydro-2′′-deoxyguanosine (8-oxodGuo) after the incubation of 2′′-deoxyguanosine (dGuo) with ChOOH and Cu2++. In the presence of copper ions, ChOOH induced DNA strand breaks in time and concentration-dependent manners. Purine and pyrimidine base modifications were also observed, as assessed respectively by the treatment with Fpg and Endo III repair enzymes. The detection of 8-oxodGuo by HPLC/MS/MS is in agreement with the dGuo oxidation in plasmid DNA. ChOOH-derived DNA damage adds further support to the role of lipid peroxidation in inducing DNA modifications and mutation. [ABSTRACT FROM AUTHOR]

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 andN1-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. [ABSTRACT FROM AUTHOR]

Decomposition of lipid hydroperoxides (LOOH) is known to generate toxic products capable to induce tissue injury. We have recently confirmed that decomposition of LOOH into peroxyl radicals is a potential source of singlet oxygen (^1O_2) in biological system. 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 ^1O_2 (^{18}[^1O_2]) by chemical trapping of ^1O_2 with 9,10-diphenylanthracene (DPA) and detecting the corresponding ^{18}O-labeled DPA endoperoxide (DPA^{18}O^{18}O) by HPLC coupled to tandem mass spectrometry (HPLC-MS/MS). ^{18}O-Labeled alcohol and ketone were also detected providing further evidence for the generation of ^1O_2by the Russell mechanism. Similarly the reaction of LA^{18}O^{18}OH with peroxynitrite also generated^{18}[^1O_2].In conclusion, these results indicates that the use of ^{18}O-labeled LOOH associated with HPLC-MS/MS can be an useful tool to clarify mechanistic features involved in the reaction of LOOH in biological media. [ABSTRACT FROM AUTHOR]

Melatonin ( N-acetyl-5-methoxytryptamine) is implicated in physiologic changes related to light–dark cycles and has been recently found to display antioxidant properties. It is known that the reaction of melatonin with certain reactive oxygen and nitrogen species, such as hydrogen peroxide and singlet oxygen, produces N1-acetyl- N2-formyl-5-methoxykynuramine (AFMK). We report herein on the development of a new liquid chromatography/tandem mass spectrometry (LC/ESI/MS-MS) assay to quantitatively determine melatonin and AFMK. The stable isotopic internal standard of melatonin-D3 was synthesized by the reaction of 5-methoxytryptamine with deuterated acetyl chloride (CD3COCl). Labeled AFMK (AFMK-D3) was obtained after photooxidation of melatonin-D3. The predominant ion [M + H]+ in the full scan mass spectra of melatonin, melatonin-D3, AFMK and AFMK-D3 were located, respectively, at m/ z = 233, 236, 265 and 268. The collision-induced dissociation of the molecules revealed a predominant fragment at m/ z = 174 for melatonin and melatonin-D3 (loss of the N-acetyl group), and at m/ z = 178 for AFMK and AFMK-D3 (loss of both the N-acetyl and the N-formyl groups). The m/ z transitions from 233 to 174 (melatonin), from 236 to 174 (melatonin-D3), from 265 to 178 (AFMK), and from 268 to 178 (AFMK-D3) were therefore chosen for the multiple reaction monitoring detection experiments, ensuring a high specificity and an accurate quantification of melatonin and AFMK in human plasma. [ABSTRACT FROM AUTHOR]

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 water-soluble 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 alkali-labile sites or oxidised pyrimidines, detectable by the bacterial endonuclease III, were not produced. [ABSTRACT FROM AUTHOR]

It has been shown that melatonin exhibits antioxidant properties. Chemical structures of some of the products formed by the interaction of melatonin with reactive oxygen and nitrogen species have been elucidated. Despite some evidence that the reaction of melatonin with singlet molecular oxygen (O2 (1 Δg )) produces N 1 -acetyl-N 2 -formyl-5-methoxykynurenine (AFMK), it has not been fully documented. In this investigation, melatonin was oxidized by photosensitization with methylene blue or by a clean chemical source of O2 (1 Δg ), the thermodecomposition of N ,N ′-di(2,3-dihydroxypropyl)-1,4-naphtalenedipropanamide (DHPNO2 ). The resulting product was characterized by high performance liquid chromatography, coupled to electrospray ionization mass spectrometry and also by 1 H, 13 C and dept135 nuclear magnetic resonance spectroscopy. An isotopically labeled DHPN18 O2 was also prepared and used as a chemical source of labeled 18 [O2 (1 Δg )] to unequivocally characterize the end product. The results uncovered by this work confirm the hypothesis that oxidation of melatonin by O2 (1 Δg ) produces AFMK. [ABSTRACT FROM AUTHOR]

Reports on the synthesis of the [[sup 18]O]-labeled endoperoxide of N,N'-di(2,3-dihydroxypropyl)-1,4-naphthalenedipropanamide. Ability of the endoperoxide to release labeled singlet oxygen; Mass spectrometry of the reaction products; Solubility in aqueous media; Mechanistic features of the reaction.

Modified oligonucleotides that contain the hydrophobic 1-hexanol-1,N6-etheno-2′-deoxyadenosine adduct have been synthesized using a mild solid phase phosphoramidite chemistry. The presence and the integrity of the modified nucleoside in the synthetic oligomers were confirmed by electrospray ionization and MALDI mass spectrometry measurements together with analysis of the complete enzymatic hydrolysate by high performance liquid chromatography coupled to UV and fluorescent detection techniques. [Copyright &y& Elsevier]