Publications by authors named "Paolo Di Mascio"

133 Publications

Detection of DNA Adduct Formation in Rat Lungs by a Micro-HPLC/MS/MS Approach.

Methods Mol Biol 2021 ;2279:225-239

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, SP, Brazil.

Aldehydes are abundantly present in tobacco smoke and in urban air pollution and are endogenously generated as products of the lipid peroxidation process. These molecules can react with DNA bases forming mutagenic exocyclic adducts, which have been used as biomarkers of aldehyde exposure and as potential tools for the study of inflammation, metal storage diseases, neurodegenerative disorders, and cancer. High-performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS) provides a highly precise, specific and ultrasensitive method for the detection of exocyclic DNA adducts. Here we present and describe a validated micro-HPLC-Electro Spray Ionization (ESI)-MS/MS method for the quantification of 1,N-propanodGuo, an adduct produced following the reaction between 2'-deoxyguanosine and acetaldehyde or crotonaldehyde.
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http://dx.doi.org/10.1007/978-1-0716-1278-1_18DOI Listing
April 2021

l-Tryptophan Interactions with the Horseradish Peroxidase-Catalyzed Generation of Triplet Acetone.

Photochem Photobiol 2021 Mar 11;97(2):327-334. Epub 2021 Feb 11.

Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil.

Triplet carbonyls generated by chemiexcitation are involved in typical photobiochemical processes in the absence of light. Due to their biradical nature, ultraweak light emission and long lifetime, electronically excited triplet species display typical radical reactions such as isomerization, fragmentation, cycloaddition and hydrogen abstraction. In this paper, we report chemical reactions in a set of amino acid residues induced by the isobutanal/horseradish peroxidase (IBAL/HRP) system, a well-known source of excited triplet acetone (Ac ). Accordingly, quenching of Ac by tryptophan (Trp) unveiled parallel enzyme damage and inactivation, likely explained by scavenging of IBAL tertiary radical reaction intermediate and Ac -derived 2-hydroxy-i-propyl radical. Quenching constants were calculated from Stern-Volmer plots, and the structure of radical adducts was revealed by mass spectrometry. As expected, a concurrent Schiff-type adduct was found to be one of the reaction by-products. These findings draw attention to potential structural and functional changes in enzymes involved in the electronic chemiexcitation of their products.
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http://dx.doi.org/10.1111/php.13363DOI Listing
March 2021

Heck reaction synthesis of anthracene and naphthalene derivatives as traps and clean chemical sources of singlet molecular oxygen in biological systems.

Photochem Photobiol Sci 2020 Nov;19(11):1590-1602

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, CP CP26077, CEP 05508-000, São Paulo, SP, Brazil.

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.
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http://dx.doi.org/10.1039/d0pp00153hDOI Listing
November 2020

Human cataractous lenses contain cross-links produced by crystallin-derived tryptophanyl and tyrosyl radicals.

Free Radic Biol Med 2020 11 26;160:356-367. Epub 2020 Aug 26.

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Av. Lineu Prestes 748, 05508-000, São Paulo, Brazil. Electronic address:

Protein insolubilization, cross-linking and aggregation are considered critical to the development of lens opacity in cataract. However, the information about the presence of cross-links other than disulfides in cataractous lenses is limited. A potential role for cross-links produced from tryptophanyl radicals in cataract development is suggested by the abundance of the UV light-sensitive Trp residues in crystallin proteins. Here we developed a LC-MS/MS approach to examine the presence of Trp-Trp, Trp-Tyr and Tyr-Tyr cross-links and of peptides containing Trp-2H (-2.0156 Da) in the lens of three patients diagnosed with advanced nuclear cataract. In the proteins of two of the lenses, we characterized intermolecular cross-links between βB2-Tyr-Tyr-βA3 and βB2-Trp-Tyr-βS. An additional intermolecular cross-link (βB2-Tyr-Trp-βB3) was present in the lens of the oldest patient. In the proteins of all three lenses, we characterized two intramolecular Trp-Trp cross-links (Trp-Trp in βB1 and Trp-Trp in βB2) and six peptides containing Trp -2H residues, which indicate the presence of additional Trp-Trp cross-links. Relevantly, we showed that similar cross-links and peptides with modified Trp-2H residues are produced in a time-dependent manner in bovine β-crystallin irradiated with a solar simulator. Therefore, different crystallin proteins cross-linked by crystalline-derived tryptophanyl and tyrosyl radicals are present in advanced nuclear cataract lenses and similar protein modifications can be promoted by solar irradiation even in the absence of photosensitizers. Overall, the results indicate that a role for Trp-Tyr and Trp-Trp cross-links in the development of human cataract is possible and deserves further investigation.
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http://dx.doi.org/10.1016/j.freeradbiomed.2020.08.020DOI Listing
November 2020

Singlet oxygen generation by the reaction of acrolein with peroxynitrite via a 2-hydroxyvinyl radical intermediate.

Free Radic Biol Med 2020 05 4;152:83-90. Epub 2020 Mar 4.

Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil; Departamento Ciências Exatas e da Terra, Instituto de Ciências Ambientais, Químicas e Farmacêuticas, Universidade Federal de São Paulo, Diadema, SP, Brazil. Electronic address:

Acrolein (2-propenal) is an environmental pollutant, food contaminant, and endogenous toxic by-product formed in the thermal decomposition and peroxidation of lipids, proteins, and carbohydrates. Like other α,β-unsaturated aldehydes, acrolein undergoes Michael addition of nucleophiles such as basic amino acids residues of proteins and nucleobases, triggering aging associated disorders. Here, we show that acrolein is also a potential target of the potent biological oxidant, nitrosating and nitrating agent peroxynitrite. In vitro studies revealed the occurrence of 1,4-addition of peroxynitrite (k = 6 × 10 M s, pH 7.2, 25 °C) to acrolein in air-equilibrated phosphate buffer. This is attested by acrolein concentration-dependent oxygen uptake, peroxynitrite consumption, and generation of formaldehyde and glyoxal as final products. These products are predicted to be originated from the Russell termination of OOCH=CH(OH) radical which also includes molecular oxygen at the singlet delta state (OΔ). Accordingly, EPR spin trapping studies with the 2,6-nitrosobenzene-4-sulfonate ion (DBNBS) revealed a 6-line spectrum attributable to the 2-hydroxyvinyl radical adduct. Singlet oxygen was identified by its characteristic monomolecular IR emission at 1,270 nm in deuterated buffer, which was expectedly quenched upon addition of water and sodium azide. These data represent the first report on singlet oxygen creation from a vinylperoxyl radical, previously reported for alkyl- and formylperoxyl radicals, and may contribute to better understand the adverse acrolein behavior in vivo.
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http://dx.doi.org/10.1016/j.freeradbiomed.2020.03.003DOI Listing
May 2020

Generation of Singlet Molecular Oxygen by Lipid Hydroperoxides and Nitronium Ion.

Photochem Photobiol 2020 05 4;96(3):560-569. Epub 2020 May 4.

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil.

Singlet molecular oxygen is a reactive species involved in biological oxidative processes. The major cellular targets of singlet molecular oxygen are unsaturated fatty acids in the membrane, as well as nucleic acids and proteins. The aim of this study was to investigate whether lipids and commercial hydroperoxides generate singlet molecular oxygen, in presence of nitronium and activated nitronium ion. For this purpose, monomol light emitted in the near-infrared region (λ = 1270 nm) was used to monitor singlet molecular oxygen decay in different solvents, with different hydroperoxides and in the presence of azide. Direct measurements of the singlet molecular oxygen spectrum at 1270 nm recorded during the reaction between lipids and commercial hydroperoxides and nitronium ions unequivocally demonstrated the formation of this excited species.
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http://dx.doi.org/10.1111/php.13236DOI Listing
May 2020

Comparing Data-Independent Acquisition and Parallel Reaction Monitoring in Their Abilities To Differentiate High-Density Lipoprotein Subclasses.

J Proteome Res 2020 01 7;19(1):248-259. Epub 2019 Nov 7.

Departamento de Bioquímica , Instituto de Química, Universidade de São Paulo , São Paulo 05513970 , Brazil.

High-density lipoprotein (HDL) is a diverse group of particles with multiple cardioprotective functions. HDL proteome follows HDL particle complexity. Many proteins were described in HDL, but consistent quantification of HDL protein cargo is still a challenge. To address this issue, the aim of this work was to compare data-independent acquisition (DIA) and parallel reaction monitoring (PRM) methodologies in their abilities to differentiate HDL subclasses through their proteomes. To this end, we first evaluated the analytical performances of DIA and PRM using labeled peptides in pooled digested HDL as a biological matrix. Next, we compared the quantification capabilities of the two methodologies for 24 proteins found in HDL and HDL from 19 apparently healthy subjects. DIA and PRM exhibited comparable linearity, accuracy, and precision. Moreover, both methodologies worked equally well, differentiating HDL subclasses' proteomes with high precision. Our findings may help to understand HDL functional diversity.
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http://dx.doi.org/10.1021/acs.jproteome.9b00511DOI Listing
January 2020

Singlet oxygen-induced protein aggregation: Lysozyme crosslink formation and nLC-MS/MS characterization.

J Mass Spectrom 2019 Nov;54(11):894-905

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil.

Singlet molecular oxygen ( O ) has been associated with a number of physiological processes. Despite the recognized importance of O -mediated protein modifications, little is known about the role of this oxidant in crosslink formation and protein aggregation. Thus, using lysozyme as a model, the present study sought to investigate the involvement of O in crosslink formation. Lysozyme was photochemically oxidized in the presence of rose bengal or chemically oxidized using [ O]-labeled O released from thermolabile endoperoxides. It was concluded that both O generating systems induce lysozyme crosslinking and aggregation. Using SDS-PAGE and nano-scale liquid chromatography coupled to electrospray ionization mass spectrometry, the results clearly demonstrated that O is directly involved in the formation of covalent crosslinks involving the amino acids histidine, lysine, and tryptophan.
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http://dx.doi.org/10.1002/jms.4448DOI Listing
November 2019

(5')-and (5')-purine 5',8-cyclo-2'-deoxyribonucleosides: reality or artifactual measurements? A reply to Chatgilialoglu's comments (this issue).

Free Radic Res 2019 Oct 25;53(9-10):1014-1018. Epub 2019 Sep 25.

Département de Médecine Nucléaire et Radiobiologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke , Sherbrooke , Canada.

This rebuttal letter is aimed at refuting the poor and false arguments elaborated by Chatgilialoglu (preceding article) in his response to the position article (Cadet et al. Free Radic Res 2019;53:574-577) that focussed on the putative reliability of the HPLC-MS/MS measurements of five radiation-induced damage to cellular DNA, which included 8-oxo-7,8-dihydro-2'-deoxyadenosine and the (5') and (5') diastereomers of 5',8-cyclo-2'-deoxyadenosine and 5',8-cyclo-2'-deoxyadenosine (Krokidis et al. Free Radic Res 2017;51:470-482). Unfortunately, none of the main issues we raised on the suitability of the analytical approach and the shortcomings associated with DNA extraction in HPLC based measurement methods of oxidatively generated damage in cells were properly considered in Chatigilialolu's letter. The main questionable issues include the lack of information on the sensitivity of HPLC-MS/MS analysis, the absence of a dose curve that is essential in the formation of damage and the nonconsideration of artifactual oxidation.
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http://dx.doi.org/10.1080/10715762.2019.1667992DOI Listing
October 2019

Quantification of three DNA Lesions by Mass Spectrometry and Assessment of Their Levels in Tissues of Mice Exposed to Ambient Fine Particulate Matter.

J Vis Exp 2019 05 29(147). Epub 2019 May 29.

Departamento de Análises Clínicas e Toxicológicas, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo;

DNA adducts and oxidized DNA bases are examples of DNA lesions that are useful biomarkers for the toxicity assessment of substances that are electrophilic, generate reactive electrophiles upon biotransformation, or induce oxidative stress. Among the oxidized nucleobases, the most studied one is 8-oxo-7,8-dihydroguanine (8-oxoGua) or 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), a biomarker of oxidatively induced base damage in DNA. Aldehydes and epoxyaldehydes resulting from the lipid peroxidation process are electrophilic molecules able to form mutagenic exocyclic DNA adducts, such as the etheno adducts 1,N-etheno-2'-deoxyguanosine (1,N-εdGuo) and 1,N-etheno-2'-deoxyadenosine (1,N-εdAdo), which have been suggested as potential biomarkers in the pathophysiology of inflammation. Selective and sensitive methods for their quantification in DNA are necessary for the development of preventive strategies to slow down cell mutation rates and chronic disease development (e.g., cancer, neurodegenerative diseases). Among the sensitive methods available for their detection (high performance liquid chromatography coupled to electrochemical or tandem mass spectrometry detectors, comet assay, immunoassays, P-postlabeling), the most selective are those based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-ESI-MS/MS). Selectivity is an essential advantage when analyzing complex biological samples and HPLC-ESI-MS/MS evolved as the gold standard for quantification of modified nucleosides in biological matrices, such as DNA, urine, plasma and saliva. The use of isotopically labeled internal standards adds the advantage of corrections for molecule losses during the DNA hydrolysis and analyte enrichment steps, as well as for differences of the analyte ionization between samples. It also aids in the identification of the correct chromatographic peak when more than one peak is present. We present here validated sensitive, accurate and precise HPLC-ESI-MS/MS methods that were successfully applied for the quantification of 8-oxodGuo, 1,N-dAdo and 1,N-dGuo in lung, liver and kidney DNA of A/J mice for the assessment of the effects of ambient PM2.5 exposure.
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http://dx.doi.org/10.3791/59734DOI Listing
May 2019

, an Endophyte That Establishes a Nutrient-Transfer Symbiosis With Banana Plants and Protects Against the Black Sigatoka Pathogen.

Front Microbiol 2019 7;10:804. Epub 2019 May 7.

Department of Chemistry, Universidad Autónoma de Guadalajara, Zapopan, Mexico.

Banana ( spp.) is an important crop worldwide, but black Sigatoka disease caused by the fungus threatens fruit production. In this work, we examined the potential of the endophytes of banana plants and , as antagonists of and support plant growth in nutrient limited soils by N-transfer. The two bacterial isolates were identified by MALDI-TOF mass spectrometry and corroborated by 16S rRNA sequence analysis. Both bacteria were positive for beneficial traits such as N-fixation, indole acetic acid production, phosphate solubilization, negative for 1-aminocyclopropane 1-carboxylic acid deaminase and were antagonistic to . To measure the effects on plant growth, the two plant bacteria and an strain (as non-endophyte), were inoculated weekly for 60 days as active cells (AC) and heat-killed cells (HKC) into plant microcosms without nutrients and compared to a water only treatment, and a mineral nutrients solution (MMN) treatment. Bacterial treatments increased growth parameters and prevented accelerated senescence, which was observed for water and mineral nutrients solution (MMN) treatments used as controls. Plants died after the first 20 days of being irrigated with water; irrigation with MMN enabled plants to develop some new leaves, but plants lost weight (-30%) during the same period. Plants treated with bacteria showed good growth, but AC treated plants had significantly greater biomass than the HKC. After 60 days, plants inoculated with AC showed intracellular bacteria within root cells, suggesting that a stable symbiosis was established. To evaluate the transference of organic N from bacteria into the plants, the 3 bacteria were grown with NHCl or NaNO as the nitrogen source. The N transferred from bacteria to plant tissues was measured by pheophytin isotopomer abundance. The relative abundance of the isotopomers 872.57, 873.57, 874.57, 875.57, 876.57 unequivocally demonstrated that plants acquired N atoms directly from bacterial cells, using them as a source of N, to support plant growth in restricted nutrient soils. might be a new alternative to promote growth and health of banana crops.
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http://dx.doi.org/10.3389/fmicb.2019.00804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6513882PMC
May 2019

Radiation-induced (5')-and (5')-purine 5',8-cyclo-2'-deoxyribonucleosides in human cells: a revisited analysis of HPLC-MS/MS measurements.

Free Radic Res 2019 May 7;53(5):574-577. Epub 2019 May 7.

a Département de Médecine Nucléaire et Radiobiologie, Faculté de Médecine et des Sciences de la Santé , Université de Sherbrooke , Sherbrooke , Canada.

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http://dx.doi.org/10.1080/10715762.2019.1605169DOI Listing
May 2019

Singlet molecular oxygen regulates vascular tone and blood pressure in inflammation.

Nature 2019 02 13;566(7745):548-552. Epub 2019 Feb 13.

Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, Australia.

Singlet molecular oxygen (O) has well-established roles in photosynthetic plants, bacteria and fungi, but not in mammals. Chemically generated O oxidizes the amino acid tryptophan to precursors of a key metabolite called N-formylkynurenine, whereas enzymatic oxidation of tryptophan to N-formylkynurenine is catalysed by a family of dioxygenases, including indoleamine 2,3-dioxygenase 1. Under inflammatory conditions, this haem-containing enzyme is expressed in arterial endothelial cells, where it contributes to the regulation of blood pressure. However, whether indoleamine 2,3-dioxygenase 1 forms O and whether this contributes to blood pressure control have remained unknown. Here we show that arterial indoleamine 2,3-dioxygenase 1 regulates blood pressure via formation of O. We observed that in the presence of hydrogen peroxide, the enzyme generates O and that this is associated with the stereoselective oxidation of L-tryptophan to a tricyclic hydroperoxide via a previously unrecognized oxidative activation of the dioxygenase activity. The tryptophan-derived hydroperoxide acts in vivo as a signalling molecule, inducing arterial relaxation and decreasing blood pressure; this activity is dependent on Cys42 of protein kinase G1α. Our findings demonstrate a pathophysiological role for O in mammals through formation of an amino acid-derived hydroperoxide that regulates vascular tone and blood pressure under inflammatory conditions.
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http://dx.doi.org/10.1038/s41586-019-0947-3DOI Listing
February 2019

Singlet Molecular Oxygen Reactions with Nucleic Acids, Lipids, and Proteins.

Chem Rev 2019 02 5;119(3):2043-2086. Epub 2019 Feb 5.

Département de Médecine Nucléaire et Radiobiologie, Faculté de Médecine des Sciences de la Santé , Université de Sherbrooke , Sherbrooke , J1H 5N4 Québec , Canada.

Singlet oxygen (O) 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 O 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 [O]-labeled thermolabile endoperoxides as a source of [O]-labeled O has been applied to study mechanistic aspects and preferential targets of O in biological systems. A relevant major topic deals with the search for the molecular signature of the O formation in targeted biomolecules within cells. It may be anticipated that [O]-labeled O and labeled peroxides in association with sensitive mass spectrometric methods should constitute powerful tools for this purpose.
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http://dx.doi.org/10.1021/acs.chemrev.8b00554DOI Listing
February 2019

Genotoxic and epigenotoxic effects in mice exposed to concentrated ambient fine particulate matter (PM) from São Paulo city, Brazil.

Part Fibre Toxicol 2018 10 19;15(1):40. Epub 2018 Oct 19.

Departamento de Análises Clínicas e Toxicológicas, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, Av. Prof. Lineu Prestes 580, Bloco 13 B, São Paulo, CEP 05508-000, Brazil.

Background: The Metropolitan Area of São Paulo has a unique composition of atmospheric pollutants, and positive correlations between exposure and the risk of diseases and mortality have been observed. Here we assessed the effects of ambient fine particulate matter (PM) on genotoxic and global DNA methylation and hydroxymethylation changes, as well as the activities of antioxidant enzymes, in tissues of AJ mice exposed whole body to ambient air enriched in PM, which was concentrated in a chamber near an avenue of intense traffic in São Paulo City, Brazil.

Results: Mice exposed to concentrated ambient PM (1 h daily, 3 months) were compared to in situ ambient air exposed mice as the study control. The concentrated PM exposed group presented increased levels of the oxidized nucleoside 8-oxo-7,8-dihydro-2'-deoxyguanosine in lung and kidney DNA and increased levels of the etheno adducts 1,N-etheno-2'-deoxyadenosine and 1,N-etheno-2'-deoxyguanosine in kidney and liver DNA, respectively. Apart from the genotoxic effects, the exposure to PM led to decreased levels of the epigenetic mark 5-hydroxymethylcytosine (5-hmC) in lung and liver DNA. Changes in lung, liver, and erythrocyte antioxidant enzyme activities were also observed. Decreased glutathione reductase and increased superoxide dismutase (SOD) activities were observed in the lungs, while the liver presented increased glutathione S-transferase and decreased SOD activities. An increase in SOD activity was also observed in erythrocytes. These changes are consistent with the induction of local and systemic oxidative stress.

Conclusions: Mice exposed daily to PM at a concentration that mimics 24-h exposure to the mean concentration found in ambient air presented, after 3 months, increased levels of DNA lesions related to the occurrence of oxidative stress in the lungs, liver, and kidney, in parallel to decreased global levels of 5-hmC in lung and liver DNA. Genetic and epigenetic alterations induced by pollutants may affect the genes committed to cell cycle control, apoptosis, and cell differentiation, increasing the chance of cancer development, which merits further investigation.
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http://dx.doi.org/10.1186/s12989-018-0276-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6194750PMC
October 2018

Exercise and β-alanine supplementation on carnosine-acrolein adduct in skeletal muscle.

Redox Biol 2018 09 18;18:222-228. Epub 2018 Jul 18.

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil. Electronic address:

Previous studies have demonstrated that exercise results in reactive aldehyde production and that β-alanine supplementation increases carnosine content in skeletal muscle. However, little is known about the influence exercise and β-alanine supplementation have on the formation of carnosine-aldehydes. The goal of the present study was to monitor the formation of carnosine-aldehyde adducts, following high-intensity intermittent exercise, before and after β-alanine supplementation. Vastus lateralis biopsy samples were taken from 14 cyclists, before and after a 28 day β-alanine supplementation, following 4 bouts of a 30 s all-out cycling test, and carnosine and CAR-aldehyde adducts [carnosine-acrolein, CAR-ACR (m/z 303), carnosine-4-hydroxy-2-hexenal, CAR-HHE (m/z 341) and carnosine-4-hydroxy-2-nonenal, CAR-HNE (m/z 383)] were quantified by HPLC-MS/MS. β-alanine supplementation increased muscle carnosine content by ~50% (p = 0.0001 vs. Pre-Supplementation). Interestingly, there was a significant increase in post-exercise CAR-ACR content following β-alanine supplementation (p < 0.001 vs. post-exercise before supplementation), whereas neither exercise alone nor supplementation alone increased CAR-ACR formation. These results suggest that carnosine functions as an acrolein-scavenger in skeletal muscle. Such a role would be relevant to the detoxification of this aldehyde formed during exercise, and appears to be enhanced by β-alanine supplementation. These novel findings not only have the potential of directly benefiting athletes who engage in intensive training regimens, but will also allow researchers to explore the role of muscle carnosine in detoxifying reactive aldehydes in diseases characterized by abnormal oxidative stress.
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http://dx.doi.org/10.1016/j.redox.2018.07.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6077140PMC
September 2018

Photosensitized Membrane Permeabilization Requires Contact-Dependent Reactions between Photosensitizer and Lipids.

J Am Chem Soc 2018 08 23;140(30):9606-9615. Epub 2018 Jul 23.

Departamento de Bioquímica, Instituto de Química , Universidade de São Paulo , Avenida Prof. Lineu Prestes 748 , São Paulo , SP , Brazil , 05508-000.

Although the general mechanisms of lipid oxidation are known, the chemical steps through which photosensitizers and light permeabilize lipid membranes are still poorly understood. Herein we characterized the products of lipid photooxidation and their effects on lipid bilayers, also giving insight into their formation pathways. Our experimental system was designed to allow two phenothiazinium-based photosensitizers (methylene blue, MB, and DO15) to deliver the same amount of singlet oxygen molecules per second to 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine liposome membranes, but with a substantial difference in terms of the extent of direct physical contact with lipid double bonds; that is, DO15 has a 27-times higher colocalization with ω-9 lipid double bonds than MB. Under this condition, DO15 permeabilizes membranes at least 1 order of magnitude more efficiently than MB, a result that was also valid for liposomes made of polyunsaturated lipids. Quantification of reaction products uncovered a mixture of phospholipid hydroperoxides, alcohols, ketones, and aldehydes. Although both photosensitizers allowed the formation of hydroperoxides, the oxidized products that require direct reactions between photosensitizer and lipids were more prevalent in liposomes oxidized by DO15. Membrane permeabilization was always connected with the presence of lipid aldehydes, which cause a substantial decrease in the Gibbs free energy barrier for water permeation. Processes depending on direct contact between photosensitizers and lipids were revealed to be essential for the progress of lipid oxidation and consequently for aldehyde formation, providing a molecular-level explanation of why membrane binding correlates so well with the cell-killing efficiency of photosensitizers.
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http://dx.doi.org/10.1021/jacs.8b05014DOI Listing
August 2018

DNA Adduct Formation in the Lungs and Brain of Rats Exposed to Low Concentrations of [C]-Acetaldehyde.

Chem Res Toxicol 2018 05 9;31(5):332-339. Epub 2018 May 9.

Departamento de Bioquímica, Instituto de Química , Universidade de São Paulo , São Paulo , SP 05508-020 , Brazil.

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 [C]-acetaldehyde induces DNA modification with the formation of isotopically labeled 1, N-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 [C]-1, N-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 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.
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http://dx.doi.org/10.1021/acs.chemrestox.8b00016DOI Listing
May 2018

Oxidation of 1-N2-etheno-2'-deoxyguanosine by singlet molecular oxygen results in 2'-deoxyguanosine: a pathway to remove exocyclic DNA damage?

Biol Chem 2018 07;399(8):859-867

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo (USP), Av. Prof. Lineu Prestes, 748, CEP 05508-000, Brazil.

Exocyclic DNA adducts are considered as potential tools for the study of oxidative stress-related diseases, but an important aspect is their chemical reactivity towards oxidant species. We report here the oxidation of 1-N2-etheno-2'-deoxyguanosine (1,N2-εdGuo) by singlet molecular oxygen (1O2) generated by a non-ionic water-soluble endoperoxide [N,N'-di(2,3-dihydroxypropyl)-1,4-naphthalenedipropanamide endoperoxide (DHPNO2)] and its corresponding oxygen isotopically labeled [18O]-[N,N'-di(2,3-dihydroxypropyl)-1,4- naphthalenedipropanamide endoperoxide (DHPN18O2)], and by photosensitization with two different photosensitizers [methylene blue (MB) and Rose Bengal (RB)]. Products detection and characterization were achieved using high performance liquid chromatography (HPLC) coupled to ultraviolet and electrospray ionization (ESI) tandem mass spectrometry, and nuclear magnetic resonance (NMR) analyses. We found that dGuo is regenerated via reaction of 1O2 with the ε-linkage, and we propose a dioxetane as an intermediate, which cleaves and loses the aldehyde groups as formate residues, or alternatively, it generates a 1,2-ethanediol adduct. We also report herein the quenching rate constants of 1O2 by 1,N2-εdGuo and other etheno modified nucleosides. The rate constant (kt) values obtained for etheno nucleosides are comparable to the kt of dGuo. From these results, we suggest a possible role of 1O2 in the cleanup of etheno adducts by regenerating the normal base.
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http://dx.doi.org/10.1515/hsz-2017-0337DOI Listing
July 2018

Lysozyme oxidation by singlet molecular oxygen: Peptide characterization using [ O]-labeling oxygen and nLC-MS/MS.

J Mass Spectrom 2017 Nov;52(11):739-751

Departamento de Bioquímica and Departamento de Química Fundamental Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil.

Singlet molecular oxygen ( O ) is generated in biological systems and reacts with different biomolecules. Proteins are a major target for O , and His, Tyr, Met, Cys, and Trp are oxidized at physiological pH. In the present study, the modification of lysozyme protein by O was investigated using mass spectrometry approaches. The experimental findings showed methionine, histidine, and tryptophan oxidation. The experiments were achieved using [ O]-labeled O released from thermolabile endoperoxides in association with nano-scale liquid chromatography coupled to electrospray ionization mass spectrometry. The structural characterization by nLC-MS/MS of the amino acids in the tryptic peptides of the proteins showed addition of [ O]-labeling atoms in different amino acids.
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http://dx.doi.org/10.1002/jms.3983DOI Listing
November 2017

In-vivo electrochemical monitoring of HO production induced by root-inoculated endophytic bacteria in Agave tequilana leaves.

Biosens Bioelectron 2018 Jan 18;99:108-114. Epub 2017 Jul 18.

Department of Fundamental Chemistry, Institute of Chemistry, University of São Paulo, Av. Prof. Lineu Prestes, 748, 05508-000 São Paulo, SP, Brazil. Electronic address:

A dual-function platinum disc microelectrode sensor was used for in-situ monitoring of HO produced in A. tequilana leaves after inoculation of their endophytic bacteria (Enterobacter cloacae). Voltammetric experiments were carried out from 0.0 to -1.0V, a potential range where HO is electrochemically reduced. A needle was used to create a small cavity in the upper epidermis of A. tequilana leaves, where the fabricated electrochemical sensor was inserted by using a manual three-dimensional micropositioner. Control experiments were performed with untreated plants and the obtained electrochemical results clearly proved the formation of HO in the leaves of plants 3h after the E. cloacae inoculation, according to a mechanism involving endogenous signaling pathways. In order to compare the sensitivity of the microelectrode sensor, the presence of HO was detected in the root hairs by 3,3-diaminobenzidine (DAB) stain 72h after bacterial inoculation. In-situ pH measurements were also carried out with a gold disc microelectrode modified with a film of iridium oxide and lower pH values were found in A. tequilana leaves treated with bacteria, which may indicate the plant produces acidic substances by biosynthesis of secondary metabolites. This microsensor could be an advantageous tool for further studies on the understanding of the mechanism of HO production during the plant-endophyte interaction.
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http://dx.doi.org/10.1016/j.bios.2017.07.039DOI Listing
January 2018

Experimental and DFT Computational Insight into Nitrosamine Photochemistry-Oxygen Matters.

J Phys Chem A 2017 Aug 2;121(32):5954-5966. Epub 2017 Aug 2.

Department of Chemistry, Brooklyn College , 2900 Bedford Avenue, Brooklyn, New York 11210, United States.

A nitrosamine photooxidation reaction is shown to generate a peroxy intermediate by experimental physical-organic methods. The irradiation of phenyl and methyl-substituted nitrosamines in the presence of isotopically labeled 18-oxygen revealed that an O atom was trapped from a peroxy intermediate to trimethylphosphite or triphenylphosphine, or by nitrosamine itself, forming two moles of nitramine. The unstable peroxy intermediate can be trapped at low temperature in postphotolyzed solution in the dark. Chemiluminescence was also observed upon thermal decomposition of the peroxy intermediate, that is, when a postphotolysis low-temperature solution is brought up to room temperature. A DFT study provides tentative information for cyclic nitrogen peroxide species on the reaction surface.
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http://dx.doi.org/10.1021/acs.jpca.7b02414DOI Listing
August 2017

Mechanism and color modulation of fungal bioluminescence.

Sci Adv 2017 Apr 26;3(4):e1602847. Epub 2017 Apr 26.

Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, Moscow 117997, Russia.

Bioluminescent fungi are spread throughout the globe, but details on their mechanism of light emission are still scarce. Usually, the process involves three key components: an oxidizable luciferin substrate, a luciferase enzyme, and a light emitter, typically oxidized luciferin, and called oxyluciferin. We report the structure of fungal oxyluciferin, investigate the mechanism of fungal bioluminescence, and describe the use of simple synthetic α-pyrones as luciferins to produce multicolor enzymatic chemiluminescence. A high-energy endoperoxide is proposed as an intermediate of the oxidation of the native luciferin to the oxyluciferin, which is a pyruvic acid adduct of caffeic acid. Luciferase promiscuity allows the use of simple α-pyrones as chemiluminescent substrates.
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http://dx.doi.org/10.1126/sciadv.1602847DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5406138PMC
April 2017

Direct participation of DNA in the formation of singlet oxygen and base damage under UVA irradiation.

Free Radic Biol Med 2017 07 18;108:86-93. Epub 2017 Mar 18.

Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, 05508-000 São Paulo, SP, Brazil. Electronic address:

UVA light is hardly absorbed by the DNA molecule, but recent works point to a direct mechanism of DNA lesion by these wavelengths. UVA light also excite endogenous chromophores, which causes DNA damage through ROS. In this study, DNA samples were irradiated with UVA light in different conditions to investigate possible mechanisms involved in the induction of DNA damage. The different types of DNA lesions formed after irradiation were determined through the use of endonucleases, which recognize and cleave sites containing oxidized bases and cyclobutane pyrimidine dimers (CPDs), as well as through antibody recognition. The formation of 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxodG) was also studied in more detail using electrochemical detection. The results show that high NaCl concentration and concentrated DNA are capable of reducing the induction of CPDs. Moreover, concerning damage caused by oxidative stress, the presence of sodium azide and metal chelators reduce their induction, while deuterated water increases the amounts of oxidized bases, confirming the involvement of singlet oxygen in the generation of these lesions. Curiously, however, high concentrations of DNA also enhanced the formation of oxidized bases, in a reaction that paralleled the increase in the formation of singlet oxygen in the solution. This was interpreted as being due to an intrinsic photosensitization mechanism, depending directly on the DNA molecule to absorb UVA and generate singlet oxygen. Therefore, the DNA molecule itself may act as a chromophore for UVA light, locally producing a damaging agent, which may lead to even greater concerns about the deleterious impact of sunlight.
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http://dx.doi.org/10.1016/j.freeradbiomed.2017.03.018DOI Listing
July 2017

Type I and Type II Photosensitized Oxidation Reactions: Guidelines and Mechanistic Pathways.

Photochem Photobiol 2017 07 27;93(4):912-919. Epub 2017 Mar 27.

Centro de Lasers e Aplicações, Instituto de Pesquisas Energéticas e Nucleares, IPEN-CNEN/SP, São Paulo, Brazil.

Here, 10 guidelines are presented for a standardized definition of type I and type II photosensitized oxidation reactions. Because of varied notions of reactions mediated by photosensitizers, a checklist of recommendations is provided for their definitions. Type I and type II photoreactions are oxygen-dependent and involve unstable species such as the initial formation of radical cation or neutral radicals from the substrates and/or singlet oxygen ( O ∆ ) by energy transfer to molecular oxygen. In addition, superoxide anion radical (O2·-) can be generated by a charge-transfer reaction involving O or more likely indirectly as the result of O -mediated oxidation of the radical anion of type I photosensitizers. In subsequent reactions, O2·- may add and/or reduce a few highly oxidizing radicals that arise from the deprotonation of the radical cations of key biological targets. O2·- can also undergo dismutation into H O , the precursor of the highly reactive hydroxyl radical (·OH) that may induce delayed oxidation reactions in cells. In the second part, several examples of type I and type II photosensitized oxidation reactions are provided to illustrate the complexity and the diversity of the degradation pathways of mostly relevant biomolecules upon one-electron oxidation and singlet oxygen reactions.
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http://dx.doi.org/10.1111/php.12716DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5500392PMC
July 2017

Sustained kidney biochemical derangement in treated experimental diabetes: a clue to metabolic memory.

Sci Rep 2017 01 12;7:40544. Epub 2017 Jan 12.

Department of Clinical and Toxicological Analyses, Faculty of Pharmaceutical Sciences, University of São Paulo, Av. Prof. Lineu Prestes 580, Bloco 13 B, CEP 05508-000, São Paulo, Brazil.

The occurrence of biochemical alterations that last for a long period of time in diabetic individuals even after adequate handling of glycemia is an intriguing phenomenon named metabolic memory. In this study, we show that a kidney pathway is gradually altered during the course of diabetes and remains persistently changed after late glycemic control in streptozotocin-induced diabetic rats. This pathway comprises an early decline of uric acid clearance and pAMPK expression followed by fumarate accumulation, increased TGF-β expression, reduced PGC-1α expression, and downregulation of methylation and hydroxymethylation of mitochondrial DNA. The sustained decrease of uric acid clearance in treated diabetes may support the prolonged kidney biochemical alterations observed after tight glycemic control, and this regulation is likely mediated by the sustained decrease of AMPK activity and the induction of inflammation. This manuscript proposes the first consideration of the possible role of hyperuricemia and the underlying biochemical changes as part of metabolic memory in diabetic nephropathy development after glycemic control.
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http://dx.doi.org/10.1038/srep40544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5228190PMC
January 2017

Formation and repair of oxidatively generated damage in cellular DNA.

Free Radic Biol Med 2017 06 2;107:13-34. Epub 2017 Jan 2.

Département de médecine nucléaire et radiobiologie, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4.

In this review article, emphasis is placed on the critical survey of available data concerning modified nucleobase and 2-deoxyribose products that have been identified in cellular DNA following exposure to a wide variety of oxidizing species and agents including, hydroxyl radical, one-electron oxidants, singlet oxygen, hypochlorous acid and ten-eleven translocation enzymes. In addition, information is provided about the generation of secondary oxidation products of 8-oxo-7,8-dihydroguanine and nucleobase addition products with reactive aldehydes arising from the decomposition of lipid peroxides. It is worth noting that the different classes of oxidatively generated DNA damage that consist of single lesions, intra- and interstrand cross-links were unambiguously assigned and quantitatively detected on the basis of accurate measurements involving in most cases high performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry. The reported data clearly show that the frequency of DNA lesions generated upon severe oxidizing conditions, including exposure to ionizing radiation is low, at best a few modifications per 10 normal bases. Application of accurate analytical measurement methods has also allowed the determination of repair kinetics of several well-defined lesions in cellular DNA that however concerns so far only a restricted number of cases.
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http://dx.doi.org/10.1016/j.freeradbiomed.2016.12.049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5457722PMC
June 2017

Ohr plays a central role in bacterial responses against fatty acid hydroperoxides and peroxynitrite.

Proc Natl Acad Sci U S A 2017 01 27;114(2):E132-E141. Epub 2016 Dec 27.

Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, 05508-090, Sao Paulo, Brazil;

Organic hydroperoxide resistance (Ohr) enzymes are unique Cys-based, lipoyl-dependent peroxidases. Here, we investigated the involvement of Ohr in bacterial responses toward distinct hydroperoxides. In silico results indicated that fatty acid (but not cholesterol) hydroperoxides docked well into the active site of Ohr from Xylella fastidiosa and were efficiently reduced by the recombinant enzyme as assessed by a lipoamide-lipoamide dehydrogenase-coupled assay. Indeed, the rate constants between Ohr and several fatty acid hydroperoxides were in the 10-10 M⋅s range as determined by a competition assay developed here. Reduction of peroxynitrite by Ohr was also determined to be in the order of 10 M⋅s at pH 7.4 through two independent competition assays. A similar trend was observed when studying the sensitivities of a ∆ohr mutant of Pseudomonas aeruginosa toward different hydroperoxides. Fatty acid hydroperoxides, which are readily solubilized by bacterial surfactants, killed the ∆ohr strain most efficiently. In contrast, both wild-type and mutant strains deficient for peroxiredoxins and glutathione peroxidases were equally sensitive to fatty acid hydroperoxides. Ohr also appeared to play a central role in the peroxynitrite response, because the ∆ohr mutant was more sensitive than wild type to 3-morpholinosydnonimine hydrochloride (SIN-1 , a peroxynitrite generator). In the case of HO insult, cells treated with 3-amino-1,2,4-triazole (a catalase inhibitor) were the most sensitive. Furthermore, fatty acid hydroperoxide and SIN-1 both induced Ohr expression in the wild-type strain. In conclusion, Ohr plays a central role in modulating the levels of fatty acid hydroperoxides and peroxynitrite, both of which are involved in host-pathogen interactions.
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http://dx.doi.org/10.1073/pnas.1619659114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5240686PMC
January 2017

Chromatin associated mechanisms in base excision repair - nucleosome remodeling and DNA transcription, two key players.

Free Radic Biol Med 2017 06 20;107:159-169. Epub 2016 Dec 20.

Laboratoire de Biologie et Modélisation de la Cellule (LBMC) CNRS/ENSL/UCBL UMR 5239 and Institut NeuroMyoGène - INMG CNRS/UCBL UMR 5310, Université de Lyon, Ecole Normale Supérieure de Lyon, 69007 Lyon, France. Electronic address:

Genomic DNA is prone to a large number of insults by a myriad of endogenous and exogenous agents. The base excision repair (BER) is the major mechanism used by cells for the removal of various DNA lesions spontaneously or environmentally induced and the maintenance of genome integrity. The presence of persistent DNA damage is not compatible with life, since abrogation of BER leads to early embryonic lethality in mice. There are several lines of evidences showing existence of a link between deficient BER, cancer proneness and ageing, thus illustrating the importance of this DNA repair pathway in human health. Although the enzymology of BER mechanisms has been largely elucidated using chemically defined DNA damage substrates and purified proteins, the complex interplay of BER with another vital process like transcription or when DNA is in its natural state (i.e. wrapped in nucleosome and assembled in chromatin fiber is largely unexplored. Cells use chromatin remodeling factors to overcome the general repression associated with the nucleosomal organization. It is broadly accepted that energy-dependent nucleosome remodeling factors disrupt histones-DNA interactions at the expense of ATP hydrolysis to favor transcription as well as DNA repair. Importantly, unlike transcription, BER is not part of a regulated developmental process but represents a maintenance system that should be efficient anytime and anywhere in the genome. In this review we will discuss how BER can deal with chromatin organization to maintain genetic information. Emphasis will be placed on the following challenging question: how BER is initiated within chromatin?
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http://dx.doi.org/10.1016/j.freeradbiomed.2016.12.026DOI Listing
June 2017

Singlet molecular oxygen: Düsseldorf - São Paulo, the Brazilian connection.

Arch Biochem Biophys 2016 Apr;595:161-75

Université de Sherbrooke, Sherbrooke, Québec JIH 5N4, Canada; CEA, INAC, F-38000, Grenoble, France.

Inspired by Helmut Sies we continue the development of suitable chemical generators of (1)O2 based on the thermodissociation of naphthalene endoperoxide derivatives. The present manuscript focuses on how the use of [(18)O]-labeled endoperoxides and hydroperoxides can be applied to study mechanistic aspects related to the generation of singlet molecular oxygen and its reactions in biological systems. The peroxidation reactions of the main cellular targets including unsaturated lipids, proteins and nucleic acids have received major attention during the last three decades. Emphasis is placed in this manuscript on the description of the synthesis and the main use of [(18)O]-labeled compounds, and especially of peroxides and (1)O2, for tracer elucidation of reaction mechanisms.
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http://dx.doi.org/10.1016/j.abb.2015.11.016DOI Listing
April 2016