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A ruthenium oxide hexacyanoferrate (RuOHCF) modified electrode was developed. Hydrodynamic voltammetry was employed to demonstrate the remarkable electrocatalytic activity toward the oxidation of 2′-deoxyguanosine. The RuOHCF modified electrode was used as amperometric detector for 2′-deoxyguanosine determination in a FIA apparatus. The influence of various experimental conditions was explored for optimum analytical performance, and at these experimental conditions, the method exhibited a linear response range to 2′-deoxyguanosine extending from 3.8 to 252 μumol L-1 with detection limit of 94 nmol L-1. Applications in DNA samples were examined, and the results for detennination of 2′-deoxyguanosine were in good agreement with those obtained by HPLC analysis Studies on the kinetics of the in vitro consumption of 2′-deoxyguanosine by acetaldehyde were also performed. [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.

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.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the dysfunction and death of motor neurons through multifactorial mechanisms that remain unclear. ALS has been recognized as a multisystemic disease, and the potential role of skeletal muscle in disease progression has been investigated. Reactive aldehydes formed as secondary lipid peroxidation products in the redox processes react with biomolecules, such as DNA, proteins, and amino acids, resulting in cytotoxic effects. 4-Hydroxy-2-nonenal (HNE) levels are elevated in the spinal cord motor neurons of ALS patients, and HNE-modified proteins have been identified in the spinal cord tissue of an ALS transgenic mice model, suggesting that reactive aldehydes can contribute to motor neuron degeneration in ALS. One biological pathway of aldehyde detoxification involves conjugation with glutathione (GSH) or carnosine (Car). Here, the detection and quantification of Car, GSH, GSSG (glutathione disulfide), and the corresponding adducts with HNE, Car-HNE, and GS-HNE, were performed in muscle and liver tissues of a hSOD1 G93A ALS rat model by reverse-phase high-performance liquid chromatography coupled to electrospray ion trap tandem mass spectrometry in the selected reaction monitoring mode. A significant increase in the levels of GS-HNE and Car-HNE was observed in the muscle tissue of the end-stage ALS animals. Therefore, analyzing variations in the levels of these adducts in ALS animal tissue is crucial from a toxicological perspective and can contribute to the development of new therapeutic strategies.

Air pollution is a major environmental risk for human health. Acetaldehyde is present in tobacco smoke and vehicle exhaust. In this study, we show that [ 13 C 2 ]-acetaldehyde induces DNA modification with the formation of isotopically labeled 1, N 2 -propano-2'-deoxyguanosine adducts in the brain and lungs of rats exposed to concentrations of acetaldehyde found in the atmosphere of megacities. The adduct, with the addition of two molecules of isotopically labeled acetaldehyde [ 13 C 4 ]-1, N 2 -propano-dGuo, was detected in the lung and brain tissues of exposed rats by micro-HPLC/MS/MS. Structural confirmation of the products was unequivocally performed by nano-LC/ESI + -HRMS 3 analyses. DNA modifications induced by acetaldehyde have been regarded as a key factor in the mechanism of mutagenesis and may be involved in the cancer risks associated with air pollution.

Acetaldehyde and crotonaldehyde are genotoxic aldehydes present in tobacco smoke and vehicle exhaust. The reaction of these aldehydes with 2'-deoxyguanosine in DNA produces α-methyl-γ-hydroxy-1,N(2)-propano-2'-deoxyguanosine (1,N(2)-propanodGuo). Online HPLC-tandem mass spectrometry was utilized to accurately quantify 1,N(2)-propanodGuo in human urinary samples from 47 residents of São Paulo City (SP) and 35 residents of the rural municipality of São João da Boa Vista (SJBV) in the state of São Paulo. Significantly higher 1,N(2)-propanodGuo levels were found in the samples from SP donors than in samples from SJBV donors. Our results provide the first evidence that elevated levels of 1,N(2)-propanodGuo in urinary samples may be correlated with urban air pollution.

Acetaldehyde is an environmentally widespread genotoxic aldehyde present in tobacco smoke, vehicle exhaust and several food products. Endogenously, acetaldehyde is produced by the metabolic oxidation of ethanol by hepatic NAD-dependent alcohol dehydrogenase and during threonine catabolism. The formation of DNA adducts has been regarded as a critical factor in the mechanisms of acetaldehyde mutagenicity and carcinogenesis. Acetaldehyde reacts with 2'-deoxyguanosine in DNA to form primarily N(2)-ethylidene-2'-deoxyguanosine. The subsequent reaction of N(2)-ethylidenedGuo with another molecule of acetaldehyde gives rise to 1,N(2)-propano-2'-deoxyguanosine (1,N(2)-propanodGuo), an adduct also found as a product of the crotonaldehyde reaction with dGuo. However, adducts resulting from the reaction of more than one molecule of acetaldehyde in vivo are still controversial. In this study, the unequivocal formation of 1,N(2)-propanodGuo by acetaldehyde was assessed in human cells via treatment with [(13)C(2)]-acetaldehyde. Detection of labeled 1,N(2)-propanodGuo was performed by HPLC/MS/MS. Upon acetaldehyde exposure (703 μM), increased levels of both 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-εdGuo), which is produced from α,β-unsaturated aldehydes formed during the lipid peroxidation process, and 1,N(2)-propanodGuo were observed. The unequivocal formation of 1,N(2)-propanodGuo in cells exposed to this aldehyde can be used to elucidate the mechanisms associated with acetaldehyde exposure and cancer risk.

In mammalian membranes, cholesterol is concentrated in lipid rafts. The generation of cholesterol hydroperoxides (ChOOHs) and their decomposition products induces various types of cell damage. The decomposition of some organic hydroperoxides into peroxyl radicals is known to be a potential source of singlet molecular oxygen [O(2) ((1)Δ(g))] in biological systems. We report herein on evidence of the generation of O(2) ((1)Δ(g)) from ChOOH isomers in solution or in liposomes containing ChOOHs, which involves a cyclic mechanism from a linear tetraoxide intermediate originally proposed by Russell. Characteristic light emission at 1270 nm, corresponding to O(2) ((1)Δ(g)) monomolecular decay, was observed for each ChOOH isomer or in liposomes containing ChOOHs. Moreover, the presence of O(2) ((1)Δ(g)) was unequivocally demonstrated using the direct spectral characterization of near-infrared light emission. Using (18)O-labeled cholesterol hydroperoxide (Ch(18)O(18)OH), we observed the formation of (18)O-labeled O(2) ((1)Δ(g)) [(18)O(2) ((1)Δ(g))] by the chemical trapping of (18)O(2) ((1)Δ(g)) with 9,10-diphenylanthracene (DPA) and detected the corresponding (18)O-labeled DPA endoperoxide (DPA(18)O(18)O) and the (18)O-labeled products of the Russell mechanism using high-performance liquid chromatography coupled to tandem mass spectrometry. Photoemission properties and chemical trapping clearly demonstrate that the decomposition of Ch(18)O(18)OH generates (18)O(2) ((1)Δ(g)), which is consistent with the Russell mechanism and points to the involvement of O(2) ((1)Δ(g)) in cholesterol hydroperoxide-mediated cytotoxicity.

Exocyclic DNA adducts produced by exogenous and endogenous compounds are emerging as potential tools to study a variety of human diseases and air pollution exposure. A highly sensitive method involving online reverse-phase high performance liquid chromatography with electrospray tandem mass spectrometry detection in the multiple reaction monitoring mode and employing stable isotope-labeled internal standards was developed for the simultaneous quantification of 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-epsilondGuo) and 1,N(2)-propano-2'-deoxyguanosine (1,N(2)-propanodGuo) in DNA. This methodology permits direct online quantification of 2'-deoxyguanosine and ca. 500 amol of adducts in 100 microg of hydrolyzed DNA in the same analysis. Using the newly developed technique, accurate determinations of 1,N(2)-etheno-2'-deoxyguanosine and 1,N(2)-propano-2'-deoxyguanosine levels in DNA extracts of human cultured cells (4.01 +/- 0.32 1,N(2)-epsilondGuo/10(8) dGuo and 3.43 +/- 0.33 1,N(2)-propanodGuo/10(8) dGuo) and rat tissue (liver, 2.47 +/- 0.61 1,N(2)-epsilondGuo/10(8) dGuo and 4.61 +/- 0.69 1,N(2)-propanodGuo/10(8) dGuo; brain, 2.96 +/- 1.43 1,N(2)-epsilondGuo/10(8) dGuo and 5.66 +/- 3.70 1,N(2)-propanodGuo/10(8) dGuo; and lung, 0.87 +/- 0.34 1,N(2)-epsilondGuo/10(8) dGuo and 2.25 +/- 1.72 1,N(2)-propanodGuo/10(8) dGuo) were performed. The method described herein can be used to study the biological significance of exocyclic DNA adducts through the quantification of different adducts in humans and experimental animals with pathological conditions and after air pollution exposure.

A few years ago, it was reported that ozone is produced in human atherosclerotic arteries, on the basis of the identification of 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al and 3beta-hydroxy-5beta-hydroxy-B-norcholestane-6beta-carboxaldehyde (ChAld) as their 2,4-dinitrophenylhydrazones. The formation of endogenous ozone was attributed to water oxidation catalyzed by antibodies, with the formation of dihydrogen trioxide as a key intermediate. We now report that ChAld is also generated by the reaction of cholesterol with singlet molecular oxygen [O2 (1Delta(g))] that is produced by photodynamic action or by the thermodecomposition of 1,4-dimethylnaphthalene endoperoxide, a defined pure chemical source of O2 (1Delta(g)). On the basis of 18O-labeled ChAld mass spectrometry, NMR, light emission measurements, and derivatization studies, we propose that the mechanism of ChAld generation involves the formation of the well-known cholesterol 5alpha-hydroperoxide (5alpha-OOH) (the major product of O2 ((1)Delta(g))-oxidation of cholesterol) and/or a 1,2-dioxetane intermediate formed by O2 (1Delta(g)) attack at the Delta(5) position. The Hock cleavage of 5alpha-OOH (the major pathway) or unstable cholesterol dioxetane decomposition (a minor pathway, traces) gives a 5,6-secosterol intermediate, which undergoes intramolecular aldolization to yield ChAld. These results show clearly and unequivocally that ChAld is generated upon the reaction of cholesterol with O2 (1Delta(g)) and raises questions about the role of ozone in biological processes.

Exocyclic DNA adducts are emerging as potential new tools for the study of oxidative stress-related diseases as well as the determination of cancer etiology and cancer risk. It is important to determine whether levels of exocyclic DNA adducts reflect redox stress in vivo and what role these adducts play in human diseases. To answer these important questions, interindividual differences, tissue distribution, background levels, and repair have to be assessed. This review focuses on recent developments in the use of these adducts as possible biomarkers for disease risk related to oxidative stress and on the challenges in developing sensitive and specific methods for clinical studies.

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.

Modification of biomolecules by reactive aldehydes is believed to play a role in biological processes, including aging, atherosclerosis, and Alzheimer's disease. Here, the modification of cytochrome c promoted by trans, trans-2,4-decadienal (DDE) was investigated. Matrix-assisted laser desorption/ionization time-of-flight experiments indicated increases in the molecular weight of cytochrome c, consistent with the formation of DDE adducts. Our data show that the protein modification was time-, pH-, and DDE concentration-dependent, leading to the formation of at least six adducts after 2 h of incubation at pH 7.4. Electrospray ionization quantitative TOF mass spectrometry analysis of tryptic digests indicated that His-33, Lys-39, Lys-72, and Lys-100 were modified by DDE. These adducts could have significant effects considering that His-33, Lys-72, and Lys-100 are present in clusters of basic amino acid residues, which are believed to participate in the interaction of cytochrome c with cardiolipin in the inner mitochondrial membrane and cytochrome c oxidase. A blue shift in the cytochrome c Soret band from 409 to 406 nm was also observed after DDE reaction, indicating heme crevice opening and displacement of heme sixth ligand (Met-80) coordination in modified protein. The covalent modifications in cytochrome c could play a role in mitochondrial dysfunction associated with oxidative stress.

A recent study showed that tetrahydrofuran (THF), a widely used solvent, is carcinogenic in experimental animals. Despite its carcinogenic activity, there is a paucity of information regarding cellular toxicity, biomolecular damage, and genotoxicity induced by THF. We describe here the structural characterization of adducts produced by the reaction of oxidized THF with 2'-deoxyguanosine (dGuo-THF 1 and dGuo-THF 2), 2'-deoxyadenosine (dAdo-THF), and 2'-deoxycytidine (dCyd-THF). Adducts were isolated from in vitro reactions by reverse-phase HPLC and fully characterized on the basis of spectroscopic measurements. The stable derivatives obtained by the reduction of adducts with NaBH(4) (the case of dGuo-THF 1, dCyd-THF, and dAdo-THF) and the stable adduct dGuo-THF 2 were used as standards for optimization of chromatographic separations for adduct detection in DNA through HPLC/ESI/MS-MS. Using this methodology, we successfully detected the four adducts in calf thymus DNA reacted with oxidized THF. The present study also provides evidence that rat liver microsomal monooxigenases oxidize THF to the reactive electrophilic compounds that are able to damage the DNA molecule, as indicated by a significant increase in adduct dGuo-THF 1 level when NADPH was added to the THF/microsomes/dGuo incubation mixtures. Our data point to DNA-THF adducts as possible contributing factors to the toxicological effects of THF exposure.

The reaction of 2'-deoxyguanosine with the alpha,beta-unsaturated aldehydes trans-2-octenal, trans-2-nonenal, trans-2-decenal, trans,trans-2,4-nonadienal, and trans,trans-2,4-decadienal in THF gives rise to three novel adducts: 3-(2'-deoxy-beta-D-erythro-pentafuranosyl)-7-[3-hydroxy-1-(3-(2'-deoxy-beta-D-erythro-pentafuranosyl)-3,5-dihydro-imidazo[1,2-a]purin-9-one-7-yl)-propyl]-3,5-dihydro-imidazo[1,2-a]purin-9-one (A7) and 3-(2'-deoxy-beta-D-erythro-pentafuranosyl)-7-(tetrahydrofuran-2-yl)-3,5-dihydro-imidazo[1,2-a]purin-9-one (A8 and A9), which are not observed in the absence of THF. These adducts were isolated from in vitro reactions by reversed-phase HPLC and fully characterized on the basis of spectroscopic measurements. Adduct A7 consists of two 1,N2-etheno-2'-deoxyguanosine (1,N2-epsilon dGuo) residues linked to a hydroxy-carbon side chain; adducts A8 and A9 are interconvertible 1,N2-epsilon dGuo derivatives bearing a THF moiety. The proposed reaction mechanism involves the electrophilic attack on 1,N2-epsilon dGuo by the carbonyl of 4-hydroxy-butanal, generated via ring opening of alpha-hydroxy-THF (THF-OH), yielding adducts A8 and A9. A further combination of these adducts with another 1,N2-epsilon dGuo produces the double adduct A7. These findings demonstrate that reactions of unsaturated aldehydes in the presence of THF produce novel condensation 1,N2-epsilon dGuo-THF adducts. Further studies would indicate the relevance of these adducts in THF toxicity.

Background levels of exocyclic DNA adducts have been detected in rodent and human tissues. Several studies have focused on bifunctional electrophiles generated from lipid peroxidation as one of the endogenous sources of these lesions. We have previously shown that the reaction of 2'-deoxyguanosine (dGuo) with trans,trans-2,4-decadienal (DDE), a highly cytotoxic aldehyde generated as a product of lipid peroxidation in cell membranes, results in the formation of a number of different base derivatives. Three of these derivatives have been fully characterized as 1,N(2)-etheno-2'-deoxyguanosine adducts. In the present work, four additional adducts, designated A3-A6, were isolated from in vitro reactions by reversed-phase HPLC and fully characterized on the basis of spectroscopic measurements. Adducts A3-A6 are four diastereoisomeric 1,N(2)-hydroxyethano-2'-deoxyguanosine derivatives possessing a carbon side chain with a double bond and a hydroxyl group. The systematic name of these adducts is 6-hydroxy-3-(2'-deoxy-beta-D-erythro-pentafuranosyl)-7-((E)-1-hydroxy-oct-2-enyl)-3,5,6,7-tetrahydro-imidazo[1,2-a]purin-9-one. The proposed reaction mechanism yielding adducts A3-A6 involves DDE epoxidation at C2, followed by nucleophilic addition of the exocyclic amino group of dGuo to the C1 of the aldehyde and cyclization, via nucleophilic attack, on the C2 epoxy group by N-1. The formation of adducts A1-A6 has been investigated in acidic, neutral, and basic pH in the presence of H(2)O(2) or tert-butyl hydroperoxide. Neutral conditions, in the presence of H(2)O(2), have favored the formation of adducts A1 and A2, with minor amounts of A3-A6, which were prevalent under basic conditions. These data indicate that DDE can modify DNA bases through different oxidative pathways involving its two double bonds. It is important to structurally characterize DNA base derivatives induced by alpha,beta-unsaturated aldehydes so that the genotoxic risks associated with the lipid peroxidation process can be assessed.

We clearly demonstrate the occurrence of energy transfer between 18O2 (1Deltag) and 16O2 in the ground state (3Sigmag-) with subsequent conversion of the latter species into its singlet excited state (1Deltag) in aqueous solution. This was inferred from the results of incubation experiments involving DHPN18O2 as a chemical generator of 18O2 (1Deltag) and the water-soluble disodium salt of anthracene (EAS) used as a chemical trap of singlet oxygen. The products of the reaction were accurately analyzed by HPLC-ESI-MS.

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.

Peroxynitrite (ONOO-), a biologically active species, can induce lipid peroxidation in biological membranes, thereby leading to the formation of various hydroperoxides. We report herein on the formation of singlet molecular oxygen [O(2) ((1)Delta(g))] in the reaction of peroxynitrite with linoleic acid hydroperoxide (LAOOH) or (18)O-labeled LAOOH. The formation of O(2) ((1)Delta(g)) was characterized by (i) dimol light emission in the red spectral region (lambda > 570 nm) using a red-sensitive photomultiplier; (ii) monomol light emission in the near-infrared region (lambda = 1270 nm) with a liquid nitrogen-cooled germanium diode or a photomultiplier coupled to a monochromator; (iii) the enhacing effect of deuterium oxide on chemiluminescence intensity, as well as the quenching effect of sodium azide; and (iv) chemical trapping of O(2) ((1)Delta(g)) or (18)O-labeled O(2) ((1)Delta(g)) with the 9,10-diphenylanthracene (DPA) and detection of the corresponding DPAO(2) or (18)O-labeled DPA endoperoxide by HPLC coupled to tandem mass spectrometry. Moreover, the presence of O(2) ((1)Delta(g)) was unequivocally demonstrated by a direct spectral characterization of the near-infrared light emission attributed to the transition of O(2) ((1)Delta(g)) to the triplet ground state. For the sake of comparison, O(2) ((1)Delta(g)) deriving from the thermolysis of the endoperoxide of 1,4-dimethylnaphthalene or from the H(2)O(2)/hypochlorite and H(2)O(2)/molybdate systems were also monitored. These novel observations identified the generation of O(2) ((1)Delta(g)) in the reaction of LAOOH with peroxynitrite, suggesting a potential O(2) ((1)Delta(g))-dependent mechanism that contributes to cytotoxicity mediated by lipid hydroperoxides and peroxynitrite reactions in biological systems.

A method involving on-line reversed-phase high-performance liquid chromatography with electrospray tandem mass spectrometry detection has been developed for the analysis of 1,N(2)-etheno-2'-deoxyguanosine 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 hydrolysate. Using the newly developed technique, basal levels of 1,N(2)-etheno-2'-deoxyguanosine were determined in commercial calf thymus DNA (1.70 +/- 0.09 adducts/10(7) dGuo), in cultured mammalian cells (CV1-P) DNA (4.5 +/- 0.4 adducts/10(7) dGuo), and in untreated female rat liver DNA (5.22 +/- 1.37 adduct/10(7) dGuo). The mutagenicity of 1,N(2)-etheno-2'-deoxyguanosine had already been demonstrated by in vitro and in vivo systems. The method described here provides the first evidence of the occurrence of 1,N(2)-etheno-2'-deoxyguanosine as a basal endogenous lesion and may be usefully employed to assess the biological consequences of etheno DNA damage under normal and pathological conditions.