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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. Evidence has been accumulated for the significant implication of singlet oxygen (1O(2)), generated as the result of UVA activation of endogenous photosensitizers as porphyrins and flavins. 7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxodGuo) has been shown to be the exclusive product of the reaction of 1O(2) with the guanine moiety of cellular DNA, in contrast to the hydroxyl radical, which reacts almost indifferently with all the nucleobases and the sugar moiety of DNA. Furthermore 8-oxodGuo is also produced by other oxidants and can be used as an ubiquitous biomarker of DNA oxidation but can not be a specific marker of any particular species. The role of DNA etheno adducts in mutagenic and carcinogenic processes triggered by known occupational and environmental carcinogens has also been studied. Much interest in etheno adducts resulted from the detection of increased levels of 1,N(6)-etheno-2'-deoxyadenosine and 3,N(4)-etheno-2'-deoxycytidine in DNA from human, rat and mouse tissues under pathophysiological conditions associated with oxidative stress. A method involving on-line HPLC with electrospray tandem mass spectrometry detection has been developed for the analysis of 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-epsilondGuo) in DNA. This methodology permits direct quantification of 20 fmol (7.4 adducts/10(8) dGuo) of the etheno adduct from approximately 350 microg of crude DNA hydrolysates. This method provides the first evidence of the occurrence of 1,N(2)-epsilondGuo as a basal endogenous lesion and may be utilized to better assess the biological consequences of etheno DNA damage under normal and pathological conditions. This work addresses the importance of isotope labeling associated with mass spectrometry technique for biomolecule damage studies.

Studies have previously shown that anthracene and naphthalene derivatives serve as compounds for trapping and chemically generating singlet molecular oxygen [O2(1Δg)], respectively. Simple and efficient synthetic routes to anthracene and naphthalene derivatives are needed, for improved capture and release of O2(1Δg) in cellular environments. Because of this need, we have synthesized a dihydroxypropyl amide naphthlene endoperoxide as a O2(1Δg) donor, as well as five anthracene derivatives as O2(1Δg) acceptor. The anthracene derivatives bear dihydroxypropyl amide, ester, and sulfonate ion end groups connected to 9,10-positions by way of unsaturated (vinyl) and saturated (ethyl) bridging groups. Heck reactions were found to yield these six compounds in easy-to-carry out 3-step reactions in yields of 50–76%. Preliminary results point to the potential of the anthracene compounds to serve as O2(1Δg) acceptors and would be amenable for future use in biological systems to expand the understanding of O2(1Δg) in biochemistry. [ABSTRACT FROM AUTHOR]

Singlet molecular oxygen O2(1Δg) is a potent oxidant that can react with different biomolecules, including DNA, lipids and proteins. Many polycyclic aromatic hydrocarbons have been studied as O2(1Δg) chemical traps. Nevertheless, a suitable modification in the polycyclic aromatic ring must be made to increase the yield of O2(1Δg) chemical trapping. With this goal, an anthracene derivative, diethyl-3,3′-(9,10-anthracenediyl)bisacrylate (DADB), was obtained from the reaction of 9,10-dibromoanthracene and ethyl acrylate through the Heck coupling reaction. The coupling of ethyl acrylate with the anthracene ring produced a new lipophilic, esterified, fluorescent probe reactive toward O2(1Δg). This compound reacts with O2(1Δg) at a rate of kr= 1.69 × 106M−1s−1forming a stable endoperoxide (DADBO2), which was characterized by UV-Vis, fluorescence, HPLC/MS and 1H and 13C NMR techniques. The photophysical, photochemical and thermostability features of DADB were also evaluated. Furthermore, this compound has the potential for great application in biological systems because it is easily synthetized in large amount and generates specific endoperoxide (DADBO2), which can be easily detected by HPLC tandem mass spectrometry (HPLC/MS/MS). [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]

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]

Singlet oxygen in its first excited state, denoted as O2, can be produced by type II photosensitization reaction and chemically in the reaction of hydrogen peroxide with hypochlorite or peroxynitrite, in the self-reaction of peroxyl radicals by the Russell mechanism, and through decomposition of dioxetanes and endoperoxides. It is also generated enzymatically by peroxidases and oxidases. The article focuses on the energy transfer between singlet and triplet molecular aqueous solution.