RESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the dysfunction and death of motor neurons through multifactorial mechanisms that remain unclear. ALS has been recognized as a multisystemic disease, and the potential role of skeletal muscle in disease progression has been investigated. Reactive aldehydes formed as secondary lipid peroxidation products in the redox processes react with biomolecules, such as DNA, proteins, and amino acids, resulting in cytotoxic effects. 4-Hydroxy-2-nonenal (HNE) levels are elevated in the spinal cord motor neurons of ALS patients, and HNE-modified proteins have been identified in the spinal cord tissue of an ALS transgenic mice model, suggesting that reactive aldehydes can contribute to motor neuron degeneration in ALS. One biological pathway of aldehyde detoxification involves conjugation with glutathione (GSH) or carnosine (Car). Here, the detection and quantification of Car, GSH, GSSG (glutathione disulfide), and the corresponding adducts with HNE, Car-HNE, and GS-HNE, were performed in muscle and liver tissues of a hSOD1G93A ALS rat model by reverse-phase high-performance liquid chromatography coupled to electrospray ion trap tandem mass spectrometry in the selected reaction monitoring mode. A significant increase in the levels of GS-HNE and Car-HNE was observed in the muscle tissue of the end-stage ALS animals. Therefore, analyzing variations in the levels of these adducts in ALS animal tissue is crucial from a toxicological perspective and can contribute to the development of new therapeutic strategies.
Assuntos
Aldeídos , Esclerose Lateral Amiotrófica , Carnosina , Modelos Animais de Doenças , Glutationa , Animais , Esclerose Lateral Amiotrófica/metabolismo , Aldeídos/metabolismo , Aldeídos/química , Carnosina/metabolismo , Glutationa/metabolismo , Ratos , Músculo Esquelético/metabolismo , Humanos , Superóxido Dismutase/metabolismo , Masculino , Cromatografia Líquida de Alta Pressão , Ratos Transgênicos , Superóxido Dismutase-1/metabolismo , Ratos Sprague-DawleyRESUMO
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 ß-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.
Assuntos
Esclerose Lateral Amiotrófica , Doenças Neurodegenerativas , Ratos , Humanos , Animais , Camundongos , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Oxilipinas , Espectrometria de Massas , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Inflamação , Modelos Animais de Doenças , Camundongos Transgênicos , Superóxido Dismutase/genéticaRESUMO
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,N2-propanodGuo, an adduct produced following the reaction between 2'-deoxyguanosine and acetaldehyde or crotonaldehyde.
Assuntos
Adutos de DNA/metabolismo , Dano ao DNA , Pulmão/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Animais , Cromatografia Líquida de Alta Pressão , Desoxiguanosina/metabolismo , RatosRESUMO
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.
Assuntos
Antracenos/farmacologia , Naftalenos/farmacologia , Oxigênio Singlete/metabolismo , Antracenos/síntese química , Antracenos/química , Linhagem Celular Tumoral , Humanos , Microscopia de Fluorescência , Estrutura Molecular , Naftalenos/síntese química , Naftalenos/química , Imagem Óptica , Oxigênio Singlete/químicaRESUMO
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 (LogPcalc > 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.
Assuntos
Esclerose Lateral Amiotrófica , Superóxido Dismutase , Aldeídos , Esclerose Lateral Amiotrófica/genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lipídeos , Mutação , Superóxido Dismutase/genética , Superóxido Dismutase-1/genéticaRESUMO
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.
Assuntos
Peróxido de Hidrogênio/química , Peróxidos Lipídicos/química , Dióxido de Nitrogênio/química , Oxigênio Singlete/química , Íons , Solventes/química , Espectroscopia de Luz Próxima ao InfravermelhoRESUMO
8-oxo-7,8-dihydroguanine, commonly referred to as 8-oxoG, is considered one of the most predominant oxidative lesions formed in DNA. Due to its ability to pair with adenines in its syn configuration, this lesion has a strong mutagenic potential in both eukaryotes and prokaryotes. Escherichia coli cells are endowed with the GO system, which protects them from the mutagenic properties of this lesion when formed both in cellular DNA and the nucleotide pool. MutY and MutM (Fpg) DNA glycosylases are crucial components of the GO system. A strong mutator phenotype of the Escherichia coli mutM mutY double mutant underscores the importance of 8-oxoG repair for genomic stability. Here, we report that in Caulobacter crescentus, a widely studied alpha-proteobacterium with a GC-rich genome, the combined lack of MutM and MutY glycosylases produces a more modest mutator phenotype when compared to E. coli. Genetic analysis indicates that other glycosylases and other repair pathways do not act synergistically with the GO system for spontaneous mutation prevention. We also show that there is not a statistically significant difference in the spontaneous levels 8-oxodGuo in E. coli and C. crescentus, suggesting that other yet to be identified differences in repair or replication probably account for the differential importance of the GO system between these two species. Environ. Mol. Mutagen. 61:246-255, 2020. © 2019 Wiley Periodicals, Inc.
Assuntos
Proteínas de Bactérias/genética , Caulobacter crescentus/genética , DNA Glicosilases/genética , DNA-Formamidopirimidina Glicosilase/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Mutagênese , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/metabolismo , DNA Glicosilases/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA-Formamidopirimidina Glicosilase/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Instabilidade Genômica , Guanina/análogos & derivados , Guanina/metabolismoRESUMO
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.
Assuntos
Aminoácidos/química , Reagentes de Ligações Cruzadas/química , Muramidase/química , Agregados Proteicos , Oxigênio Singlete/química , Alquilação , Cromatografia Líquida de Alta Pressão , Oxirredução , Processos Fotoquímicos , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
Amyotrophic lateral sclerosis (ALS) is characterized by progressive loss of upper and lower motor neurons leading to muscle paralysis and death. While a link between dysregulated lipid metabolism and ALS has been proposed, lipidome alterations involved in disease progression are still understudied. Using a rodent model of ALS overexpressing mutant human Cu/Zn-superoxide dismutase gene (SOD1-G93A), we performed a comparative lipidomic analysis in motor cortex and spinal cord tissues of SOD1-G93A and WT rats at asymptomatic (~70 days) and symptomatic stages (~120 days). Interestingly, lipidome alterations in motor cortex were mostly related to age than ALS. In contrast, drastic changes were observed in spinal cord of SOD1-G93A 120d group, including decreased levels of cardiolipin and a 6-fold increase in several cholesteryl esters linked to polyunsaturated fatty acids. Consistent with previous studies, our findings suggest abnormal mitochondria in motor neurons and lipid droplets accumulation in aberrant astrocytes. Although the mechanism leading to cholesteryl esters accumulation remains to be established, we postulate a hypothetical model based on neuroprotection of polyunsaturated fatty acids into lipid droplets in response to increased oxidative stress. Implicated in the pathology of other neurodegenerative diseases, cholesteryl esters appear as attractive targets for further investigations.
Assuntos
Esclerose Lateral Amiotrófica/patologia , Metabolismo dos Lipídeos/genética , Neurônios Motores/metabolismo , Medula Espinal/patologia , Superóxido Dismutase-1/genética , Envelhecimento/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Cardiolipinas/análise , Cardiolipinas/metabolismo , Ésteres do Colesterol/análise , Ésteres do Colesterol/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Ácidos Graxos Insaturados/análise , Ácidos Graxos Insaturados/metabolismo , Feminino , Humanos , Gotículas Lipídicas/patologia , Lipidômica , Masculino , Espectrometria de Massas , Córtex Motor/metabolismo , Neurônios Motores/química , Mutação , Estresse Oxidativo/genética , Ratos , Ratos Transgênicos , Medula Espinal/química , Medula Espinal/citologia , Medula Espinal/metabolismo , Superóxido Dismutase-1/metabolismoRESUMO
Singlet oxygen (1O2) 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 1O2 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 [18O]-labeled thermolabile endoperoxides as a source of [18O]-labeled 1O2 has been applied to study mechanistic aspects and preferential targets of 1O2 in biological systems. A relevant major topic deals with the search for the molecular signature of the 1O2 formation in targeted biomolecules within cells. It may be anticipated that [18O]-labeled 1O2 and labeled peroxides in association with sensitive mass spectrometric methods should constitute powerful tools for this purpose.
Assuntos
Lipídeos/química , Ácidos Nucleicos/química , Proteínas/química , Oxigênio Singlete/química , Animais , Humanos , Metabolismo dos Lipídeos , Ácidos Nucleicos/metabolismo , Proteínas/metabolismo , Oxigênio Singlete/metabolismoRESUMO
ß-alanine is the rate-limiting point for the endogenous synthesis of carnosine in skeletal muscle. Carnosine has a wide range of implications for health, normal function and exercise performance. Whilst the physiological relevance of carnosine to different tissues remains enigmatic, ß-alanine administration is a useful strategy to investigate the physiological roles of carnosine in humans. Intravenous administration of ß-alanine is an interesting approach to study carnosine metabolism. However, sterilisation is mandatory due to the nature of the administration route. We evaluated whether sterilising doses of gamma radiation damages the molecular structure and leads to the loss of functional characteristics of ß-alanine. Pure ß-alanine was sterilised by gamma radiation in sealed glass vials using a 60Co multipurpose irradiator at a dose rate of 8.5 kGy.hour-1 totalising 10, 20, 25 30 and 40 kGy. The molecular integrity was assessed by X-ray Diffraction and changes in content were determined by High Performance Liquid Chromatography (UV-HPLC) and Triple Quadrupole Mass Spectrometer (HPLC/MS-MS). Sterility assurance was evaluated by inoculation assay. To examine whether functional properties were preserved, ß-alanine was infused in one participant, who rated the level of paraesthesia on the skin using a 0-3 scale. Urinary ß-alanine was quantified before and 24-h following ß-alanine infusion using HPLC-ESI+-MS/MS. Irradiation resulted in no change in the crystal structure of ß-alanine, no degradation, and no new peaks were identified in the dose range assayed. The inoculation assay showed the absence of viable microorganisms in all ß-alanine samples, including those that did not undergo irradiation. Intravenous infusion of ß-alanine resulted in paraesthesia and it detected in the urine as per normal. We conclude that gamma radiation is a suitable technique for the sterilisation of ß-alanine. It does not lead to degradation, damage to the ß-alanine structure, content or loss of function within the evaluated irradiation conditions.
Assuntos
Raios gama , beta-Alanina/química , Cromatografia Líquida de Alta Pressão , Humanos , Estrutura Molecular , Músculo Esquelético/metabolismo , Músculo Esquelético/efeitos da radiação , Difração de Raios X , beta-Alanina/metabolismoRESUMO
In C. crescentus, iron metabolism is mainly controlled by the transcription factor Fur (ferric uptake regulator). Iron-bound Fur represses genes related to iron uptake and can directly activate the expression of genes for iron-containing proteins. In this work, we used total RNA sequencing (RNA-seq) of wild type C. crescentus growing in minimal medium under iron limitation and a fur mutant strain to expand the known Fur regulon, and to identify novel iron-regulated genes. The RNA-seq of cultures treated with the iron chelator 2-2-dypiridyl (DP) allowed identifying 256 upregulated genes and 236 downregulated genes, being 176 and 204 newly identified, respectively. Sixteen transcription factors and seven sRNAs were upregulated in iron limitation, suggesting that the response to low iron triggers a complex regulatory network. Notably, lexA along with most of its target genes were upregulated, suggesting that DP treatment caused DNA damage, and the SOS DNA repair response was activated in a RecA-dependent manner, as confirmed by RT-qPCR. Fluorescence microscopy assays using an oxidation-sensitive dye showed that wild type cells in iron limitation and the fur mutant were under endogenous oxidative stress, and a direct measurement of cellular H2O2 showed that cells in iron-limited media present a higher amount of endogenous H2O2. A mutagenesis assay using the rpoB gene as a reporter showed that iron limitation led to an increase in the mutagenesis rate. These results showed that iron deficiency causes C. crescentus cells to suffer oxidative stress and to activate the SOS response, indicating an increase in DNA damage.
RESUMO
Previous studies have demonstrated that exercise results in reactive aldehyde production and that ß-alanine supplementation increases carnosine content in skeletal muscle. However, little is known about the influence exercise and ß-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 ß-alanine supplementation. Vastus lateralis biopsy samples were taken from 14 cyclists, before and after a 28â¯day ß-alanine supplementation, following 4 bouts of a 30â¯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. ß-alanine supplementation increased muscle carnosine content by ~50% (pâ¯=â¯0.0001 vs. Pre-Supplementation). Interestingly, there was a significant increase in post-exercise CAR-ACR content following ß-alanine supplementation (pâ¯<â¯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 ß-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.
Assuntos
Acroleína/metabolismo , Carnosina/metabolismo , Suplementos Nutricionais , Treinamento Intervalado de Alta Intensidade , Músculo Esquelético/fisiologia , beta-Alanina/metabolismo , Adulto , Aldeídos/metabolismo , Método Duplo-Cego , Humanos , Estresse OxidativoRESUMO
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 [13C2]-acetaldehyde induces DNA modification with the formation of isotopically labeled 1, N2-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 [13C4]-1, N2-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+-HRMS3 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.
Assuntos
Acetaldeído/toxicidade , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Adutos de DNA/biossíntese , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Acetaldeído/administração & dosagem , Acetaldeído/química , Animais , Isótopos de Carbono , Adutos de DNA/química , Adutos de DNA/isolamento & purificação , Masculino , Estrutura Molecular , Ratos , Ratos WistarRESUMO
Singlet molecular oxygen (1 O2 ) is generated in biological systems and reacts with different biomolecules. Proteins are a major target for 1 O2 , and His, Tyr, Met, Cys, and Trp are oxidized at physiological pH. In the present study, the modification of lysozyme protein by 1 O2 was investigated using mass spectrometry approaches. The experimental findings showed methionine, histidine, and tryptophan oxidation. The experiments were achieved using [18 O]-labeled 1 O2 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 [18 O]-labeling atoms in different amino acids.
Assuntos
Muramidase/química , Peptídeos/química , Oxigênio Singlete/química , Aminoácidos/química , Cromatografia Líquida de Alta Pressão/métodos , Oxirredução , Isótopos de Oxigênio , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
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 Ca2+ 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 Ca2+ 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 Ca2+ handling related to elevated intramitochondrial ATP.
Assuntos
Cálcio/metabolismo , Restrição Calórica , Fígado/metabolismo , Mitocôndrias Hepáticas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Trifosfato de Adenosina/metabolismo , Animais , Peróxido de Hidrogênio/metabolismo , Fígado/patologia , Masculino , Camundongos , Poro de Transição de Permeabilidade Mitocondrial , NAD/metabolismo , Consumo de Oxigênio/fisiologia , Permeabilidade , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologiaRESUMO
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-ß 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.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Rim/metabolismo , Rim/patologia , Adenilato Quinase/metabolismo , Animais , Glicemia/metabolismo , DNA Mitocondrial/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Jejum/sangue , Fumaratos/metabolismo , Hiperglicemia/sangue , Hiperglicemia/fisiopatologia , Rim/fisiopatologia , Masculino , Malondialdeído/metabolismo , Mitocôndrias/metabolismo , Modelos Biológicos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Fosforilação , Ratos Wistar , Fator de Crescimento Transformador beta/metabolismoRESUMO
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.
Assuntos
Peróxidos/química , Oxigênio Singlete , BrasilRESUMO
Aldehydes accumulate in inflammation, during myocardial infarction and have been associated with pain symptoms. One pathway of aldehyde detoxification is the conjugation with carnosine. A 3-methylpyridinium carnosine adduct from the reaction of carnosine and acrolein was characterized using extensive spectroscopic measurements. The adduct with urinary concentrations of 1.82 ± 0.68 nmol/mg of creatinine is one of the most abundant acrolein metabolites in urine and opens promising therapeutic strategies for carnosine.