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Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive loss of motor neurons, systemic hypermetabolism, and inflammation. In this context, oxylipins have been investigated as signaling molecules linked to neurodegeneration, although their specific role in ALS remains unclear. Importantly, most methods focused on oxylipin analysis are based on low-resolution mass spectrometry, which usually confers high sensitivity, but not great accuracy for molecular characterization, as provided by high-resolution MS (HRMS). Here, we established an ultra-high performance liquid chromatography HRMS (LC-HRMS) method for simultaneous analysis of 126 oxylipins in plasma. Intra- and inter-day method validation showed high sensitivity (0.3-25 pg), accuracy and precision for more than 90% of quality controls. This method was applied in plasma of ALS rats overexpressing the mutant human Cu/Zn-superoxide dismutase gene (SOD1-G93A) at asymptomatic (ALS 70 days old) and symptomatic stages (ALS 120 days old), and their respective age-matched wild type controls. From the 56 oxylipins identified in plasma, 17 species were significantly altered. Remarkably, most of oxylipins linked to inflammation and oxidative stress derived from arachidonic acid (AA), like prostaglandins and mono-hydroxides, were increased in ALS 120 d rats. In addition, ketones derived from AA and linoleic acid (LA) were increased in both WT 120 d and ALS 120 d groups, supporting that age also modulates oxylipin metabolism in plasma. Interestingly, the LA-derived diols involved in fatty acid uptake and β-oxidation, 9(10)-DiHOME and 12(13)-DiHOME, were decreased in ALS 120 d rats and showed significant synergic effects between age and disease factors. In summary, we validated a high-throughput LC-HRMS method for oxylipin analysis and provided a comprehensive overview of plasma oxylipins involved in ALS disease progression. Noteworthy, the oxylipins altered in plasma have potential to be investigated as biomarkers for inflammation and hypermetabolism in ALS., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Unsaturated lipids are oxidized by reactive oxygen species and enzymes, leading to the increased formation of lipid hydroperoxides and several electrophilic products. Lipid-derived electrophiles can modify macromolecules, such as proteins, resulting in the loss of function and/or aggregation. The accumulation of Cu,Zn-superoxide dismutase (SOD1) aggregates has been associated with familial cases of amyotrophic lateral sclerosis (ALS). The protein aggregation mechanisms in motor neurons remain unclear, although recent studies have shown that lipids and oxidized lipid derivatives may play roles in this process. Here, we aimed to compare the effects of different lipid aldehydes on the induction of SOD1 modifications and aggregation, in vitro. Human recombinant apo-SOD1 was incubated with 4-hydroxy-2-hexenal (HHE), 4-hydroxy-2-nonenal (HNE), 2-hexen-1-al (HEX), 2,4-nonadienal (NON), 2,4-decadienal (DEC), or secosterol aldehydes (SECO-A or SECO-B). High-molecular-weight apo-SOD1 aggregates dramatically increased in the presence of highly hydrophobic aldehydes (LogP calc  > 3). Notably, several Lys residues were modified by exposure to all aldehydes. The observed modifications were primarily observed on Lys residues located near the dimer interface (K3 and K9) and at the electrostatic loop (K122, K128, and K136). Moreover, HHE and HNE induced extensive apo-SOD1 modifications, by forming Schiff bases or Michael adducts with Lys, His, and Cys residues. However, these aldehydes were unable to induce large protein aggregates. Overall, our data shed light on the importance of lipid aldehyde hydrophobicity on the induction of apo-SOD1 aggregation and identified preferential sites of lipid aldehyde-induced modifications., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Caloric restriction (CR) promotes lifespan extension and protects against many pathological conditions, including ischemia/reperfusion injury to the brain, heart and kidney. In the liver, ischemia/reperfusion damage is related to excessive mitochondrial Ca 2+ accumulation, leading to the mitochondrial permeability transition. Indeed, liver mitochondria isolated from animals maintained on CR for 4 months were protected against permeability transition and capable of taking up Ca 2+ at faster rates and in larger quantities. These changes were not related to modifications in mitochondrial respiratory activity, but rather to a higher proportion of ATP relative to ADP in CR liver mitochondria. Accordingly, both depletion of mitochondrial adenine nucleotides and loading mitochondria with exogenous ATP abolished the differences between CR and ad libitum (AL) fed groups. The prevention against permeability transition promoted by CR strongly protected against in vivo liver damage induced by ischemia/reperfusion. Overall, our results show that CR strongly protects the liver against ischemia/reperfusion and uncover a mechanism for this protection, through a yet undescribed diet-induced change in liver mitochondrial Ca 2+ handling related to elevated intramitochondrial ATP., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Ultraweak chemiluminescence arising from biomolecules oxidation has been attributed to the radiative deactivation of singlet molecular oxygen [(1)O2] and electronically excited triplet carbonyl products involving dioxetane intermediates. As examples, we will discuss the generation of (1)O2 from lipid hydroperoxides, which involves a cyclic mechanism from a linear tetraoxide intermediate. The generation of (1)O2 in aqueous solution via energy transfer from the excited triplet acetone arising from the thermodecomposition of dioxetane a chemical source, and horseradish peroxidase-catalyzed oxidation of 2-methylpropanal, as an enzymatic source, will also be discussed. The approach used to unequivocally demonstrate the generation of (1)O2 in these reactions is the use of (18)O-labeled hydroperoxide / triplet dioxygen ((18)[(3)O2]), the detection of labeled compounds by HPLC coupled to tandem mass spectrometry (HPLC-MS/MS) and the direct spectroscopic detection and characterization of (1)O2 light emission. Characteristic light emission at 1,270nm, corresponding to the singlet delta state monomolecular decay was observed. Using(18)[(3)O2], we observed the formation of (18)O-labeled (1)O2 ((18)[(1)O2]) by the chemical trapping of (18)[(1)O2]with the anthracene-9,10-diyldiethane-2,1-diyl disulfate disodium salt (EAS) and detected the corresponding (18)O-labeled EAS endoperoxide usingHPLC-MS/MS. The combined use of the thermolysis of a water-soluble naphthalene endoperoxide as a generator of (18)O labeled (1)O2 and the sensitivity of HPLC-MS/MS allowed the study of (1)O2reactivity toward biomolecules. Photoemission properties and chemical trapping clearly demonstrate that the production of hydroperoxide and excited carbonyls generates (18)[(1)O2], and points to the involvement of (1)O2 in physiological and pathophysiological mechanism. Supported by FAPESP (2012/12663-1), CAPES, INCT Redoxoma (FAPESP/CNPq/CAPES; 573530/2008-4), NAP Redoxoma (PRPUSP; 2011.1.9352.1.8), CEPID Redoxoma (FAPESP; 2013/07937-8)., (Copyright © 2014. Published by Elsevier Inc.)

Air pollution is a major risk for human health. Acetaldehyde is an environmental pollutant present in tobacco smoke, vehicle exhaust and several food products. 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 primarily form N(2)-ethylidene-2'-deoxyguanosine (N(2)-ethylidene-dGuo). The subsequent reaction of N(2)-ethylidene-dGuo with another molecule of acetaldehyde gives rise to 1,N(2)-propano-2´-deoxyguanosine (1,N(2)-propanodGuo). In this study, on-line reverse-phase high-performance liquid chromatography (HPLC) separation with tandem mass spectrometry detection was utilized for the accurate quantification of 1,N(2)-propanodGuo and 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-edGuo) in tissues of rats exposed to 12 ppb, 33 ppb and 96 ppb acetaldehyde in atmospheric air for 50 days. A significant increase in the levels of 1,N(2)-propanodGuo was observed in lung tissues of rats exposed to 12 ppb (7.8/10(8) dGuo); 33 ppb (8.9/10(8) dGuo) and 96 ppb (11.6/10(8) dGuo) compared to controls (4.2/10(8) dGuo). For comparative purposes, the levels of 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-edGuo), which is produced from a,b-unsaturated aldehydes formed during the lipid peroxidation process were also measured. Elevated levels of 1,N(2)-edGuo were observed only in lung tissues of animals exposed to 96 ppb acetaldehyde. 1,N(2)-propanodGuo also differed quantitatively in liver but not in brain. The monitoring of 1,N(2)-propanodGuo levels in tissues provides important information on acetaldehyde genotoxicity and may contribute to the elucidation of the mechanisms associated with acetaldehyde exposure and cancer risk. Supported byFAPESP:2011/10048-5, CAPES, INCT Redoxoma:573530/2008-4,NAP Redoxoma: 2011.1.9352.1.8, CEPID Redoxoma:2013/07937-8., (Copyright © 2014. Published by Elsevier Inc.)

Calorie restriction is a dietary intervention known to improve redox state, glucose tolerance, and animal life span. Other interventions have been adopted as study models for caloric restriction, including nonsupplemented food restriction and intermittent, every-other-day feedings. We compared the short- and long-term effects of these interventions to ad libitum protocols and found that, although all restricted diets decrease body weight, intermittent feeding did not decrease intra-abdominal adiposity. Short-term calorie restriction and intermittent feeding presented similar results relative to glucose tolerance. Surprisingly, long-term intermittent feeding promoted glucose intolerance, without a loss in insulin receptor phosphorylation. Intermittent feeding substantially increased insulin receptor nitration in both intra-abdominal adipose tissue and muscle, a modification associated with receptor inactivation. All restricted diets enhanced nitric oxide synthase levels in the insulin-responsive adipose tissue and skeletal muscle. However, whereas calorie restriction improved tissue redox state, food restriction and intermittent feedings did not. In fact, long-term intermittent feeding resulted in largely enhanced tissue release of oxidants. Overall, our results show that restricted diets are significantly different in their effects on glucose tolerance and redox state when adopted long-term. Furthermore, we show that intermittent feeding can lead to oxidative insulin receptor inactivation and glucose intolerance., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The decomposition of organic hydroperoxides into peroxyl radicals is a potential source of singlet molecular oxygen [O2 (1Deltag)] in biological systems. This study shows that 5-(hydroperoxymethyl)uracil (5-HPMU), a thymine hydroperoxide within DNA, reacts with metal ions or HOCl, generating O2 (1Deltag). Spectroscopic evidence for generation of O2 (1Deltag) was obtained by measuring (i) the bimolecular decay, (ii) the monomolecular decay, and (iii) the observation of D2O enhancement of O2 (1Deltag) production and the quenching effect of NaN3. Moreover, the presence of O2 (1Deltag) was unequivocally demonstrated by the direct characterization of the near-infrared light emission. For the sake of comparison, O2 (1Deltag) derived from the H2O2/HOCl system and from the thermolysis of the N,N'-di(2,3-dihydroxypropyl)-1,4-naphthalenedipropanamide endoperoxide was also monitored. More evidence of O2 (1Deltag) generation was obtained by chemical trapping of O2 (1Deltag) with anthracene-9,10-divinylsulfonate (AVS) and detection of the specific AVS endoperoxide by HPLC/MS/MS. The detection by HPLC/MS of 5-(hydroxymethyl)uracil and 5-formyluracil, two thymine oxidation products generated from the reaction of 5-HPMU and Ce4+ ions, supports the Russell mechanism. These photoemission properties and chemical trapping clearly demonstrate that the decomposition of 5-HPMU generates O2 (1Deltag) by the Russell mechanism and point to the involvement of O2 (1Deltag) in thymidine hydroperoxide cytotoxicity.

It is now well established that oxidation of 2'-deoxyguanosine (dGuo) in DNA by singlet molecular oxygen [O2 (1Delta(g))] produces 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), whereas the main degradation products of free dGuo in aqueous solution have been identified as the two diastereomers of spiroiminodihydantoin nucleoside. Interestingly, O2 (1Delta(g))-mediated oxidation of free 8-oxodGuo gives rise to a pattern of degradation products that is different from that observed when the nucleoside is inserted into DNA. The reasons for these differences and the mechanisms involved in the oxidation reactions are not yet completely understood for either dGuo or 8-oxodGuo, either free or within DNA. In the present work, we report a study of the reaction of O2 (1Delta(g)) toward a modified nucleoside, 8-methoxy-2'-deoxyguanosine (8-MeOdGuo), either free or incorporated into an oligonucleotide. The reason for the choice of 8-MeOdGuo as a chemical model to study in more detail the oxidation pathways of 8-oxodGuo or, more precisely, of the tautomeric 8-hydroxy-2'-deoxyguanosine was dictated by the fact that only the 7,8-enolic tautomer is present in the molecule. The thermolysis of an endoperoxide of a naphthalene derivative as a clean chemical source of 18O-labeled O2 (1Delta(g)) was used to oxidize 8-MeOdGuo. The main O2 (1Delta(g)) oxidation products that were separated and analyzed by HPLC coupled to tandem mass spectrometry were identified as the 2'-deoxyribonucleoside derivatives of 2,2,4-triamino-5-(2H)oxazolone, 2,5-diamino-4H-imidazol-4-one together with the methyl-substituted derivatives of spiroiminodihydantoin, oxidized iminoallantoin and urea. On the other hand, O2 (1Delta(g)) oxidation of 8-MeOdGuo-containing oligonucleotide generated imidazolone as the predominant degradation product. These results provided new mechanistic insights into the reactions of O2 (1Delta(g)) with purine nucleosides.

Modified oligonucleotides that contain the hydrophobic 1-hexanol-1,N(6)-etheno-2'-deoxyadenosine adduct have been synthesized using a mild solid phase phosphoramidite chemistry. The presence and the integrity of the modified nucleoside in the synthetic oligomers were confirmed by electrospray ionization and MALDI mass spectrometry measurements together with analysis of the complete enzymatic hydrolysate by high performance liquid chromatography coupled to UV and fluorescent detection techniques.