Plasmalogen oxidation induces the generation of excited molecules and electrophilic lipid species.

Plasmalogens are glycerophospholipids with a vinyl ether linkage at the sn-1 position of the glycerol backbone. Despite being suggested as antioxidants due to the high reactivity of their vinyl ether groups with reactive oxygen species, our study reveals the generation of subsequent reactive oxygen and electrophilic lipid species from oxidized plasmalogen intermediates. By conducting a comprehensive analysis of the oxidation products by liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS), we demonstrate that singlet molecular oxygen [O2 (1Δg)] reacts with the vinyl ether bond, producing hydroperoxyacetal as a major primary product (97%) together with minor quantities of dioxetane (3%). Furthermore, we show that these primary oxidized intermediates are capable of further generating reactive species including excited triplet carbonyls and O2 (1Δg) as well as electrophilic phospholipid and fatty aldehyde species as secondary reaction products. The generation of excited triplet carbonyls from dioxetane thermal decomposition was confirmed by light emission measurements in the visible region using dibromoanthracene as a triplet enhancer. Moreover, O2 (1Δg) generation from dioxetane and hydroperoxyacetal was evidenced by detection of near-infrared light emission at 1,270 nm and chemical trapping experiments. Additionally, we have thoroughly characterized alpha-beta unsaturated phospholipid and fatty aldehydes by LC-HRMS analysis using two probes that specifically react with aldehydes and alpha-beta unsaturated carbonyls. Overall, our findings demonstrate the generation of excited molecules and electrophilic lipid species from oxidized plasmalogen species unveiling the potential prooxidant nature of plasmalogen-oxidized products.

Excited state properties of 5-fluoro-4-thiouridine derivative†.

The excited state properties of thionated 5-fluorouridine (2',3',5'-tri-O-acetyl-5-fluoro-4-thiouridine; ta5F4TUrd), synthesized with Lawesson's reagent, have been intensively investigated with nanosecond transient absorption spectroscopy, time-resolved thermal lensing, near-infrared emission, and quantum chemical calculation. The intrinsic triplet lifetime of ta5F4TUrd was determined to be 4.2 ± 0.7 µs in acetonitrile, and the formation quantum yield of the excited triplet state was as large as 0.79 ± 0.01 . The quenching rate constants of the triplet ta5F4TUrd by the dissolved oxygen molecule and by the self-quenching process were found to be nearly equal to the diffusion-controlled rate of acetonitrile. The quantum yield of the singlet molecular oxygen produced through energy transfer between the triplet ta5F4TUrd and the dissolved oxygen, Φ Δ , was successfully determined to be 0.61 ± 0.02 under the oxygen-saturated condition. From the oxygen concentration dependence of the Φ Δ value, the fraction of triplet ta5F4TUrd quenched by dissolved oxygen which gives rise to the 1 O2 * formation, S Δ , was successfully obtained to be 0.78 ± 0.01 , which was the largest among the thionucleobases and the thionucleosides reported so far. This could be due to the lower energy and/or the ππ* character of the triplet state.

Evaluation of Probes to Measure Oxidizing Organic Triplet Excited States in Aerosol Liquid Water.

Oxidizing triplet excited states of organic matter (3C*) drive numerous reactions in fog/cloud drops and aerosol liquid water (ALW). Quantifying oxidizing triplet concentrations in ALW is difficult because 3C* probe loss can be inhibited by the high levels of dissolved organic matter (DOM) and copper in particle water, leading to an underestimate of triplet concentrations. In addition, illuminated ALW contains high concentrations of singlet molecular oxygen (1O2*), which can interfere with 3C* probes. Our overarching goal is to find a triplet probe that has low inhibition by DOM and Cu(II) and low sensitivity to 1O2*. To this end, we tested 12 potential probes from a variety of compound classes. Some probes are strongly inhibited by DOM, while others react rapidly with 1O2*. One of the probe candidates, (phenylthiol)acetic acid (PTA), seems well suited for ALW conditions, with mild inhibition and fast rate constants with triplets, but it also has weaknesses, including a pH-dependent reactivity. We evaluated the performance of both PTA and syringol (SYR) as triplet probes in aqueous extracts of particulate matter. While PTA is less sensitive to inhibition than SYR, it results in lower triplet concentrations, possibly because it is less reactive with weakly oxidizing triplets.

NIR-II Ratiometric Chemiluminescent/Fluorescent Reporters for Real-Time Monitoring and Evaluating Cancer Photodynamic Therapy Efficacy.

The development of probes for early monitoring tumor therapy response may greatly benefit the promotion of photodynamic therapy (PDT) efficacy. Singlet oxygen (1 O2 ) generation is a typical indicator for evaluating PDT efficacy in cancer. However, most existing probes cannot quantitatively detect 1 O2 in vivo due to the high reactivity and transient state, and thus have a poor correlation with PDT response. Herein, a 1 O2 -responsive theranostic platform comprising thiophene-based small molecule (2SeFT-PEG) and photosensitizer Chlorin e6 (Ce6) micelles for real-time monitoring PDT efficacy is developed. After laser irradiation, the Ce6-produced 1 O2 could simultaneously kill cancer and trigger 2SeFT-PEG to produce increased chemiluminescence (CL) and decreased fluorescence (FL) signals variation at 1050 nm in the second near-infrared (NIR-II, 950-1700 nm) window. Significantly, the ratiometric NIR-II CL/FL imaging at 1050 nm could effectively quantify and monitor the concentration of 1 O2 and O2 consumption or recovery, so as to evaluate the therapeutic efficacy of PDT in vivo. Hence, this 1 O2 activated NIR-II CL/FL probe provides an efficient ratiometric optical imaging platform for real-time evaluating PDT effect and precisely guiding the PDT process in vivo.

Dehydromethionine is a common product of methionine oxidation by singlet molecular oxygen and hypohalous acids.

Methionine is one of the main targets for biological oxidants. Its reaction with the majority of oxidants generates only methionine sulfoxide. However, when N-terminal methionine reacts with hypohalous acids (HOCl and HOBr) or singlet molecular oxygen (1O2), it can also generate a cyclic product called dehydromethionine (DHM). Previously, DHM was suggested as a biomarker of oxidative stress induced by hypohalous acids. However, DHM can also be generated by 1O2 -oxidation of methionine, and the contribution of this pathway of DHM formation in a context of a site-specific redox imbalance in an organism is unknown. In this work, a through comparison of the reactions of hypohalous acids and 1O2 with methionine, either free or inserted in peptides and proteins was undertaken. In addition, we performed methionine photooxidation in heavy water (H218O) to determine the influence of the pH in the mechanism of DHM formation. We showed that for free methionine, or methionine-containing peptides, the yields of DHM formation in the reactions with 1O2 were close to those achieved by HOBr oxidation, but much higher than the yields obtained with HOCl as the oxidant. This was true for all pH tested (5, 7.4, and 9). Interestingly, for the protein ubiquitin, DHM yields after reaction with 1O2 were higher than those obtained with both hypohalous acids. Our results indicate that 1O2 may also be an important source of DHM in biological systems.

Haloperoxidase-Catalyzed Luminol Luminescence.

Common peroxidase action and haloperoxidase action are quantifiable as light emission from dioxygenation of luminol (5-amino-2,3-dihydrophthalazine-1,4-dione). The velocity of enzyme action is dependent on the concentration of reactants. Thus, the reaction order of each participant reactant in luminol luminescence was determined. Horseradish peroxidase (HRP)-catalyzed luminol luminescence is first order for hydrogen peroxide (H2O2), but myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are second order for H2O2. For MPO, reaction is first order for chloride (Cl-) or bromide (Br-). For EPO, reaction is first order for Br-. HRP action has no halide requirement. For MPO and EPO, reaction is first order for luminol, but for HRP, reaction is greater than first order for luminol. Haloperoxidase-catalyzed luminol luminescence requires acidity, but HRP action requires alkalinity. Unlike the radical mechanism of common peroxidase, haloperoxidases (XPO) catalyze non-radical oxidation of halide to hypohalite. That reaction is second order for H2O2 is consistent with the non-enzymatic reaction of hypohalite with a second H2O2 to produce singlet molecular oxygen (1O2*) for luminol dioxygenation. Alternatively, luminol dehydrogenation by hypohalite followed by reaction with H2O2 yields dioxygenation consistent with the same reaction order. Haloperoxidase action, Cl-, and Br- are specifically quantifiable as luminol luminescence in an acidic milieu.

A General Method to Establish the Relative Efficiency of Different Sonosensitizers to Generate ROS for SDT.

The most common way to demonstrate the reactive oxygen species (ROS)-mediated pathways in photodynamic therapy (PDT) and in sonodynamic therapy (SDT) is the use of specific ROS inhibitors. We present a general method to establish the relative efficiency of different sonosensitizers which produce the same ROS. To demonstrate it, we use peroxides as sonosensitizers which produce singlet molecular oxygen. The method is easily generalized by all types of ROS.

Metal-Free, Visible-Light-Induced Selective C-C Bond Cleavage of Cycloalkanones with Molecular Oxygen.

A metal-free, visible-light-induced oxidative C-C bond cleavage of cycloketones with molecular oxygen is described. Cooperative Brønsted-acid catalysis and photocatalysis enabled selective C-C bond cleavage of cycloketones to generate an array of γ-, δ- and ϵ-keto esters under very mild conditions. Mechanistic studies indicate that singlet molecular oxygen (1 O2 ) is responsible for this transformation.

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

Singlet molecular oxygen (1 O2 ) has been associated with a number of physiological processes. Despite the recognized importance of 1 O2 -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 1 O2 in crosslink formation. Lysozyme was photochemically oxidized in the presence of rose bengal or chemically oxidized using [18 O]-labeled 1 O2 released from thermolabile endoperoxides. It was concluded that both 1 O2 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 1 O2 is directly involved in the formation of covalent crosslinks involving the amino acids histidine, lysine, and tryptophan.

Influence of pH and micellar systems on the sensitized photo-oxidation of bovine serum albumin.

Photosensitized oxidation of bovine serum albumin (BSA), by using perinaphtenone as a sensitizer, has been studied at pH 7.4 and 11. The selected sensitizer does not present ground-state complexation with BSA and ensures that the mechanism is mediated by O2 (1 △g ). Strong dependence between BSA-O2 (1 △g ) photo-oxidation and the pH of the medium has been found. The relative oxygen uptake rate (v- â–³ O2 ) and the total quenching rate constant (kt ) values are higher at pH 11 than pH 7.4. The enhancement in the alkaline condition is due to conformational changes in the protein and the reactivity of tyrosinate anion with O2 (1 △g ). Even when the tendency with the pH in the presence of sodium dodecyl sulfate (SDS) micelles is similar to that observed in homogeneous media, an increment on the kt value is detected. This effect may be attributable to the strong interaction of BSA-SDS, which leads to the protein unfolding and could leave more exposed photo-oxidizable amino acids. A protective effect against the O2 (1 △g )-mediated photo-oxidation was observed in reverse micelles (RMs) of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) by comparing the kt values obtained at W = 10 with respect to the one obtain in homogeneous media. The latter could be mainly explained by the modification in the solvent polarity. Also, another important observation was found, the internal pH inside RMs of AOT sensed through tyrosine absorption was independent of the one used for the formation of the water pool. Hence, the kt values observed at both pH, are quite similar.

Mechanism of the O2 (1 Δg ) generation from the Cl2 /H2 O2 basic aqueous solution explored by the combined ab initio calculation and nonadiabatic dynamics simulation.

In the present work, mechanism of the O2 (1 Δg ) generation from the reaction of the dissolved Cl2 with H2 O2 in basic aqueous solution has been explored by the combined ab initio calculation and nonadiabatic dynamics simulation, together with different solvent models. Three possible pathways have been determined for the O2 (1 Δg ) generation, but two of them are sequentially downhill processes until formation of the OOCl- complex with water, which are of high exothermic character. Once the complex is formed, singlet molecular oxygen is easily generated by its decomposition along the singlet-state pathway. However, triplet molecular oxygen of O2 ( Σ 3 g - ) can be produced with considerable probability through nonadiabatic intersystem crossing in the 1 Δg / Σ 3 g - intersection region. It has been found that the coupled solvent, heavy-atom, and nonadiabatic effects have an important influence on the quantum yield of the O2 (1 Δg ) generation. © 2018 Wiley Periodicals, Inc.

Generation of ROS mediated by mechanical waves (ultrasound) and its possible applications.

The thermal decomposition of 9,10 diphenylanthracene peroxide (DPAO2) generates DPA and a mix of triplet and singlet molecular oxygen. For DPAO2 the efficiency to produce singlet molecular oxygen is 0.35. On the other hand, it has shown that many thermal reactions can be carried out through the interaction of molecules with ultrasound. Ultrasound irradiation can create hydrodynamic stress (sonomechanical process), inertial cavitation (pyrolitic process) and long range effects mediated by radicals or ROS. Sonochemical reactions can be originated by pyrolytic like process, shock mechanical waves, thermal reactions and radical and ROS mediated reactions. Sonolysis of pure water can yield hydrogen or hydroxyl radicals and hydrogen peroxide (ROS). When DPAO2 in 1,4 dioxane solution is treated with 20 or 24kHz and different power intensity the production of molecular singlet oxygen is observed. Specific scavengers like tetracyclone (TC) are used to demonstrate it. The efficiency now is 0.85 showing that the sonochemical process is much more efficient that the thermal one. Another endoperoxide, artemisinin was also studied. Unlike the concept of photosensitizer of photodynamic therapy, in spite of large amount of reported results in literature, the term sonosensitizer and the sonosensitization process are not well defined. We define sonosensitized reaction as one in which a chemical species decompose as consequence of cavitation phenomena producing ROS or other radicals and some other target species does undergo a chemical reaction. The concept could be reach rapidly other peroxides which are now under experimental studies. For artemisinin, an important antimalarian and anticancer drug, was established that ultrasound irradiation increases the effectiveness of the treatment but without any explanation. We show that artemisinin is an endoperoxide and behaves as a sonosensitizer in the sense of our definition.

Glutathione modifies the oxidation products of 2'-deoxyguanosine by singlet molecular oxygen.

The oxidation of the free nucleoside 2'-deoxyguanosine (dGuo) by singlet molecular oxygen ((1)O2) has been studied over the three last decades due to the major role of DNA oxidation products in process such as ageing, mutation and carcinogenesis. In the present work we investigated the dGuo oxidation by (1)O2 in the presence of the important low molecular antioxidant, glutathione, in its reduced (GSH) and oxidized (GSSG) forms. There were applied different conditions of concentration, pH, time of incubation, and the use of a [(18)O]-labeled thermolabile endoperoxide naphthalene derivative as a source of [(18)O]-labeled (1)O2. Data was obtained through high performance liquid chromatography (HPLC) and HPLC coupled to micrOTOF Q-II analysis of the main oxidation products: the diastereomers of spiroiminodihydantoin-2'-deoxyribonucleosides (dSp) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo). An intriguing result was that 8-oxodGuo levels increased by 100 fold when dGuo was oxidized by (1)O2 in the presence of GSH and by 2 fold in the presence of GSSG, while dSp levels dropped to zero for both conditions. All data from dGuo, 8-oxodGuo and dSp quantification together with the analysis of residual GSH/GSSG content in each sample strongly suggest that glutathione modifies the mechanism of dGuo oxidation by (1)O2 by disfavoring the pathway of dSp formation.

Photodegradation of antibiotic 5-sulfaminouracil in the presence of vitamin B2: a kinetic study.

OBJECTIVE: This paper studies the kinetics and mechanism of the sensitized photodegradation of 5-sulfaminouracil (SFU). This compound is a synthetic bacteriostatic belonging to the sulfa drugs, usually detected in surface water and effluent wastewater. METHODS: SFU interacts with electronically excited singlet and triplet states of riboflavin (Rf), (1)Rf* and (3)Rf*, respectively. The rate constants for these processes were determined in MeOH-H2O by stationary fluorescence spectroscopy (for kQ(1)) and by laser flash photolysis (for kQ(3)). RESULTS: SFU is photodegraded by visible light irradiation in the presence of the natural sensitizer (Rf). In competitive processes, (3)Rf* generates the reactive oxygen species superoxide radical anion (O2(•-)) and singlet molecular oxygen [O2((1)Δg)], which are involved in SFU photodegradation. In aqueous solutions, where SFU adopts different forms depending on the pH of the medium, the participation of O2((1)Δg) is predominant. Therefore, the O2((1)Δg)-mediated mechanism was evaluated at pHs 7 and 12, employing perinaphthenone as a synthetic photosensitizer. DISCUSSION: The results indicate that SFU is photodegraded through a relatively efficient process in a neutral environment, whereas it is quickly degraded in alkaline media. This is attributed to the ionization of the sulfamino- group, a substituent in the uracil molecule that exerts an activating power on the molecule. Thus, sensitized photodegradation may be an important tool to reduce the environmental impact of SFU.

Singlet molecular oxygen generated by biological hydroperoxides.

The chemistry behind the phenomenon of ultra-weak photon emission has been subject of considerable interest for decades. Great progress has been made on the understanding of the chemical generation of electronically excited states that are involved in these processes. Proposed mechanisms implicated the production of excited carbonyl species and singlet molecular oxygen in the mechanism of generation of chemiluminescence in biological system. In particular, attention has been focused on the potential generation of singlet molecular oxygen in the recombination reaction of peroxyl radicals by the Russell mechanism. In the last ten years, our group has demonstrated the generation of singlet molecular oxygen from reactions involving the decomposition of biologically relevant hydroperoxides, especially from lipid hydroperoxides in the presence of metal ions, peroxynitrite, HOCl and cytochrome c. In this review we will discuss details on the chemical aspects related to the mechanism of singlet molecular oxygen generation from different biological hydroperoxides.

Pharmaceutical development, composition and quantitative analysis of phthalocyanine as the photosensitizer for cancer photodynamic therapy.

Phthalocyanine (Pc) and its related derivatives are a class of functional materials that are easily activated by the light at a special wavelength. As such photosensitizer, Pc has been applied to photodynamic therapy (PDT), in addition to its broad applications in many fields, for both malignant and benign diseases. One of our long-term research focuses is to develop Pc for cancer therapy. Herein we briefly review mechanisms of action of Pc used for photodynamic therapy, its pharmaceutical development and molecular modification to enhance its drugability and improve its intracellular localization. We also describe the current status of the Pc derivatives under clinical investigation, and analyze the methods used for quantitative analysis of those Pc derivatives.

Comparisons between conventional, ultrasound-assisted and microwave-assisted methods for extraction of anthraquinones from Heterophyllaea pustulata Hook f. (Rubiaceae).

This work reports a comparative study about extraction methods used to obtain anthraquinones (AQs) from stems and leaves of Heterophyllae pustulata Hook (Rubiáceae). One of the conventional procedures used to extract these metabolites from a vegetable matrix is by successive Soxhlet extractions with solvents of increasing polarity: starting with hexane to eliminate chlorophylls and fatty components, following by benzene and finally ethyl acetate. However, this technique shows a low extraction yield of total AQs, and consumes large quantities of solvent and time. Ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE) have been investigated as alternative methods to extract these compounds, using the same sequence of solvents. It was found that UAE increases the extraction yield of total AQs and reduces the time and amount of solvent used. Nevertheless, the combination UAE with benzene, plus MAE with ethyl acetate at a constant power of 900 W showed the best results. A higher yield of total AQs was obtained in less time and using the same amount of solvent that UAE. The optimal conditions for this latter procedure were UAE with benzene at 50 °C during 60 min, followed by MAE at 900 W during 15 min using ethyl acetate as extraction solvent.

Singlet molecular oxygen-quenching activity of carotenoids: relevance to protection of the skin from photoaging.

Carotenoids are known to be potent quenchers of singlet molecular oxygen [O(2) ((1)Δ(g))]. Solar light-induced photooxidative stress causes skin photoaging by accelerating the generation of reactive oxygen species via photodynamic actions in which O(2) ((1)Δ(g)) can be generated by energy transfer from excited sensitizers. Thus, dietary carotenoids seem to participate in the prevention of photooxidative stress by accumulating as antioxidants in the skin. An in vivo study using hairless mice clarified that a O(2) ((1)Δ(g)) oxygenation-specific peroxidation product of cholesterol, cholesterol 5α-hydroperoxide, accumulates in skin lipids due to ultraviolet-A exposure. Matrix metalloproteinase-9, a metalloproteinase family enzyme responsible for the formation of wrinkles and sagging, was enhanced in the skin of ultraviolet-A -irradiated hairless mice. The activation of metalloproteinase-9 and the accumulation of 5α-hydroperoxide, as well as formation of wrinkles and sagging, were lowered in mice fed a ß-carotene diet. These results strongly suggest that dietary ß-carotene prevents the expression of metalloproteinase-9 (at least in part), by inhibiting the photodynamic action involving the formation of 5α-hydroperoxide in the skin. Intake of ß-Carotene therefore appears to be helpful in slowing down ultraviolet-A -induced photoaging in human skin by acting as a O(2) ((1)Δ(g)) quencher.

Hidroperóxidos de lipídios como fontes de oxigênio molecular singlete (O2 [1 DELTA g]), detecção e danos em biomoléculas / Lipid hidroperoxides as singlet molecular oxygen precursors (O2 [1 DELTA g]), detection and damage to biomolecules

O estudo do processo da peroxidação de lipídios tem aumentado nos últimos anos, principalmente devido à implicação dos hidroperóxidos de lipídios (LOOH) em diversos processos patológicos. A decomposição destes LOOH é capaz de gerar subprodutos capazes de promover danos em biomoléculas, incluindo proteínas e DNA. No presente trabalho, utilizando hidroperóxidos de ácido linoléico isotopicamente marcado com átomo de oxigênio-18 (LA18O18OH), fomos capazes de demonstrar que estas moléculas gerararam oxigênio singlete marcado [18(1O2)] em células em cultura. A detecção de tal espécie foi possível através da utilização de uma nova metodologia utilizando um derivado antracenico. Para este propósito foi utilizado o derivado de antraceno 3,3'-(9,10-antracenodiil) bisacrilato (DADB), cujo produto especifico da reação com o 1O2 (o endoperóxido do DADB DADBO2) do pode ser facilmente detectado por HPLC-MS/MS. De forma a expandir a compreensão dos efeitos tóxicos desses LOOH, investigamos o efeito destes compostos gerados intracelularmente. Para tal, foi utilizado o Rosa bengala (RB), um fotosensibilizador que tem afinidade por espaços apolares como membranas e lisossomos. A fotosenssibilização deste composto foi capaz de induzir a morte celular, e esta morte estaria relacionada a uma maior formação de 1O2 e a um maior acumulo de peróxidos. Nestes estudos foi possível demonstrar que carotenóides e sistemas antioxidantes dependentes de glutationa foram capazes de proteger contra os efeitos tóxicos da fotosensibilização na presença de RB. Adicionalmente foram avaliados os efeitos da hemoglobina (Hb) e do hidroperóxido do ácido linoléico (LAOOH) em uma série de parâmetros toxicológicos, como citotoxicidade, estado redox, a peroxidação lipídica e dano ao DNA. Nós demonstramos que a pré-incubação das células com Hb e sua posterior exposição à LAOOH (Hb + LAOOH) levou a um aumento na morte celular, a oxidação do DCFH, formação de malonaldeído e fragmentação do DNA e que esses...

The study of the process of lipid peroxidation has increased in recent years, mainly due to the involvement of lipid hydroperoxide (LOOH) in a series of pathological processes. The decomposition of LOOH is able to generate products that can promote damage to biomolecules, including proteins and DNA. In the present work, using linoleic acid hydroperoxide isotopically labeled with 18O2 (LA18O18OH), we demonstrate that these molecules were able to generate labeled singlet oxygen [18(1O2)] in cultured cells. The detection of such species was possible using a new methodology using an anthracene derivative .For this purpose we used the anthracene derivative of 3,3'-(9,10-antracendiil) bisacrilate (DADB), whose specific reaction product with 1O2 (DADB endoperoxide DADBO2) can be easily detected by HPLC-MS/MS. In order to expand the understanding of the toxic effects of LOOH, we investigated the effect of these compounds generated intracellularly. For this porpoise, we used Rose Bengal (RB), a photosensitizer that has affinity for apolar spaces such as membranes and lysosomes. The photosensitization of this compound was able to induce cell death, and this death was related to increased formation of 1O2 and a higher accumulation of peroxides. In these studies we have shown that carotenoids and glutathione-dependent antioxidant systems were capable of protecting against the toxic effects of photosensitization in the presence of RB. Additionally, we evaluated the effects of hemoglobin (Hb) and linoleic acid hydroperoxide (LAOOH) in a series of toxicological endpoints such as cytotoxicity, redox status, lipid peroxidation and DNA damage. We demonstrated that preincubation of cells with Hb and its subsequent exposure to LAOOH (Hb + LAOOH) led to an increase in cell death, DCFH oxidation, formation of malonaldehyde and DNA fragmentation, and that these effects were related to the peroxide and the heme group. It was demonstrated that cells incubated with LAOOH and Hb showed...

Geração de ozônio isotopicamente marcado com átomo de oxigênio-18, (18O3), formando oxigênio-18 molecular singlete, 18O2 (1[delta]g), e modificações na 2'- desoxiguanosine / Isotopically labeled ozone, 18O3, generate 18O-labeled singlet molecular oxygen, 18O2 (1[delta]g), and oxidation of product of the purine moiety of 2'-deoxyguanosine

O ozônio (O3) é um poderoso oxidante e quantidades significativas podem ser formadas em ambientes urbanos, como resultado de uma série de eventos fotoquímicos, sendo um risco para a saúde humana. Devido a sua reatividade química, o ozônio é capaz de promover modificações oxidativas em diversas biomoléculas, tais como, DNA, proteínas e lipídios. As reações do O3 com biomoléculas geram quantidades significativas de O2 (1Δg). Sendo assim, essas reações são caracterizadas pela transferência de um átomo de oxigênio do O3 ao substrato oxidado. Devido à regra de conservação do Spin, isto requer que o dioxigênio gerado nesta reação esteja no seu estado singlete. Neste específico mecanismo, a formação do hidrotrióxido tem sido frequentemente assumida como um importante intermediário da ozonização. Ainda, constatou-se o elevado potencial mutagênico do O3 sobre o DNA, levando, principalmente, à substituição de suas bases. A frequência das substituições das bases foi essencialmente localizada no par G: C's (75%), uma característica das espécies reativas de oxigênio, como o O2 (1Δg). No entanto, os mecanismos pelos quais O3 causa danos ao DNA ainda não foram completamente elucidados. No presente trabalho, as evidências espectroscópicas na geração do O2 (1Δg) foram obtidas através da emissão de luz bimolecular na região vermelha do espectro (λ = 634 nm) e através da emissão de luz monomolecular na região do infravermelho próximo (λ = 1270 nm ) durante a reação de O3 com dGuo e 8-oxodGuo. Além disso, desenvolveu-se uma metodologia para a geração de ozônio isotopicamente marcado com átomo de oxigênio-18 a partir do 18O2 (3Σg-). Deste modo, as evidências da formação dos diastereoisômeros da spiroiminodihidantoina, tanto a isotopicamente marcada no 18O quanto a não marcada, juntamente com a 8-oxodGuo, imidazolona e oxazolona, foram detectados como produtos de oxidação das reações com 18O3. Para tal observação, análises foram realizadas por HPLC acoplado...

Ozone (O3) is a potent oxidant and significant amounts can be formed in urban environments as a result of a series of complex photochemical events. It is a threat for human health. Due its chemical reactivity towards biological targets, ozone is able to promote oxidative modification in several biomolecules, such as DNA, proteins and lipids. Reactions of O3 with biomolecules are able to generate in high yields of singlet molecular oxygen [O2 (1Δg)]. The transfer of one oxygen atom from O3 to the oxidized substrate characterizes these reactions. Spin conservation rules require that the dioxygen generated in this reaction has to be in its singlet state. In this specific mechanism, hydrotrioxide has often been assumed as important intermediates in the ozonization process. In addition, ozone has been established as a powerful mutagenic agent, and the most observed mutation is in G:C transversion. This kind of transversion is typical in reactions involving DNA and reactive oxygen species, such as O2 (1Δg). However, the mechanisms by which O3 causes DNA damage have not yet been fully elucidated. In the present research, spectroscopic evidence for the generation of O2 (1Δg) was obtained by measuring the dimol light emission in the red spectral region (λ = 634 nm) and the monomol light emission in the near-infrared region (λ=1270 nm). Both measuements were done during interaction of O3 with dGuo and 8-oxodGuo. In addition, a system was built to produce isotopically labeled ozone with 18O. Thefore, in the same system that 8-oxodGuo, imidazolone and oxazolone, 18O-labeled and unlabeled diastereoisomeric spiroiminodihydantoin nucleosides were detected as the oxidation products with 18O3. In that case, analyses by HPLC coupled to mass spectrometry were performed. Moreover, in the O3 decomposition the formation of 18O-labeled O2 (1Δg) from 18O-labeled ozone was obtained by chemical trapping of O2 (1Δg) with EAS anthracene derivative and detected the corresponding...