RESUMO

Glutathione (GSH) is one of the main antioxidant molecules present in cells. It harbors a thiol group responsible for sustaining cellular redox homeostasis. This moiety can react with cellular electrophiles such as formaldehyde yielding the compound S-hydroxymethyl-GSH (HSMGSH). HSMGSH is the substrate of the enzyme alcohol dehydrogenase 5 (ADH5) and thus a key intermediate in formaldehyde metabolism. In this work, we describe a method for the chemical synthesis of HSMGSH and a pipeline to identify this compound in complex cell extracts by means of ultra-high-performance liquid chromatography coupled to high-resolution spectrometry (UHPLC-HRMS). This method also allows determining GSH and oxidized disulfide (GSSG) in the same samples, thus providing broad information about formaldehyde-GSH metabolism.

Assuntos

Antioxidantes , Glutationa , Humanos , Dissulfeto de Glutationa/química , Cromatografia Líquida de Alta Pressão/métodos , Glutationa/metabolismo , Antioxidantes/metabolismo , Compostos de Sulfidrila , Oxirredução

RESUMO

Formaldehyde (FA) is a ubiquitous endogenous and environmental metabolite that is thought to exert cytotoxicity through DNA and DNA-protein crosslinking, likely contributing to the onset of the human DNA repair condition Fanconi Anaemia. Mutations in the genes coding for FA detoxifying enzymes underlie a human inherited bone marrow failure syndrome (IBMFS), even in the presence of functional DNA repair, raising the question of whether FA causes relevant cellular damage beyond genotoxicity. Here, we report that FA triggers cellular redox imbalance in human cells and in Caenorhabditis elegans. Mechanistically, FA reacts with the redox-active thiol group of glutathione (GSH), altering the GSH:GSSG ratio and causing oxidative stress. FA cytotoxicity is prevented by the enzyme alcohol dehydrogenase 5 (ADH5/GSNOR), which metabolizes FA-GSH products, lastly yielding reduced GSH. Furthermore, we show that GSH synthesis protects human cells from FA, indicating an active role of GSH in preventing FA toxicity. These findings might be relevant for patients carrying mutations in FA-detoxification systems and could suggest therapeutic benefits from thiol-rich antioxidants like N-acetyl-L-cysteine.

Assuntos

Aldeído Oxirredutases/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Anemia de Fanconi/metabolismo , Formaldeído/toxicidade , Glutationa/metabolismo , Aldeído Oxirredutases/genética , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Dano ao DNA , Modelos Animais de Doenças , Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Formaldeído/metabolismo , Técnicas de Inativação de Genes , Células HCT116 , Humanos , Oxirredução , Estresse Oxidativo

RESUMO

AIM: Hereditary hemochromatosis (HH) is a group of inherited disorders that causes a slow and progressive iron deposition in diverse organs, particularly in the liver. Iron overload induces oxidative stress and tissue damage. Coenzyme Q10 (CoQ10) is a cofactor in the electron-transport chain of the mitochondria, but it is also a potent endogenous antioxidant. CoQ10 interest has recently grown since various studies show that CoQ10 supplementation may provide protective and safe benefits in mitochondrial diseases and oxidative stress disorders. In the present study we sought to determine CoQ10 plasma level in patients recently diagnosed with HH and to correlate it with biochemical, genetic, and histological features of the disease. METHODS: Plasma levels of CoQ10, iron, ferritin, transferrin and vitamins (A, C and E), liver tests (transaminases, alkaline phosphatase and bilirubin), and histology, as well as three HFE gene mutations (H63D, S654C and C282Y), were assessed in thirty-eight patients (32 males, 6 females) newly diagnosed with HH without treatment and in twenty-five age-matched normolipidemic healthy subjects with no HFE gene mutations (22 males, 3 females) and without clinical or biochemical signs of iron overload or liver diseases. RESULTS: Patients with HH showed a significant decrease in CoQ10 levels respect to control subjects (0.31 ±â€¯0.03 µM vs 0.70 ±â€¯0.06 µM, p < 0.001, respectively) independently of the genetic mutation, cirrhosis, transferrin saturation, ferritin level or markers of hepatic dysfunction. Although a decreasing trend in CoQ10 levels was observed in patients with elevated iron levels, no correlation was found between both parameters in patients with HH. Vitamins C and A levels showed no changes in HH patients. Vitamin E was significantly decreased in HH patients (21.1 ±â€¯1.3 µM vs 29.9 ±â€¯2.5 µM, p < 0.001, respectively), but no correlation was observed with CoQ10 levels. CONCLUSION: The decrease in CoQ10 levels found in HH patients suggests that CoQ10 supplementation could be a safe intervention strategy complementary to the traditional therapy to ameliorate oxidative stress and further tissue damage induced by iron overload.

Assuntos

Ataxia , Hemocromatose , Doenças Mitocondriais , Debilidade Muscular , Ubiquinona/deficiência , Ataxia/epidemiologia , Estudos de Casos e Controles , Feminino , Hemocromatose/sangue , Hemocromatose/epidemiologia , Hemocromatose/genética , Humanos , Masculino , Doenças Mitocondriais/epidemiologia , Debilidade Muscular/epidemiologia , Ubiquinona/análogos & derivados , Ubiquinona/sangue

RESUMO

Clear cell renal cell carcinoma (ccRCC) is a heterogeneous disease with 50-80% patients exhibiting mutations in the von Hippel-Lindau (VHL) gene. RSUME (RWD domain (termed after three major RWD-containing proteins: RING finger-containing proteins, WD-repeat-containing proteins, and yeast DEAD (DEXD)-like helicases)-containing protein small ubiquitin-related modifier (SUMO) enhancer) acts as a negative regulator of VHL function in normoxia. A discovery-based metabolomics approach was developed by means of ultraperformance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (MS) for fingerprinting the endometabolome of a human ccRCC cell line 786-O and three other transformed cell systems (n = 102) with different expressions of RSUME and VHL. Cross-validated orthogonal projection to latent structures discriminant analysis models were built on positive, negative, and a combination of positive- and negative-ion mode MS data sets. Discriminant feature panels selected by an iterative multivariate classification allowed differentiating cells with different expressions of RSUME and VHL. Fifteen identified discriminant metabolites with level 1, including glutathione, butyrylcarnitine, and acetylcarnitine, contributed to understand the role of RSUME in ccRCC. Altered pathways associated with the RSUME expression were validated by biological and bioinformatics analyses. Combined results showed that in the absence of VHL, RSUME is involved in the downregulation of the antioxidant defense system, whereas in the presence of VHL, it acts in rerouting energy-related pathways, negatively modulating the lipid utilization, and positively modulating the fatty acid synthesis, which may promote deposition in droplets.

Assuntos

Carcinoma de Células Renais , Neoplasias Renais , Carcinoma de Células Renais/genética , Linhagem Celular Tumoral , Humanos , Neoplasias Renais/genética , Espectrometria de Massas , Fatores de Transcrição , Proteína Supressora de Tumor Von Hippel-Lindau/genética

RESUMO

Exposure to ambient air particulate matter (PM) is associated with increased cardiorespiratory morbidity and mortality. In this context, alveolar macrophages exhibit proinflammatory and oxidative responses as a result of the clearance of particles, thus contributing to lung injury. However, the mechanisms linking these pathways are not completely clarified. Therefore, the oxinflammation phenomenon was studied in RAW 264.7 macrophages exposed to Residual Oil Fly Ash (ROFA), a PM surrogate rich in transition metals. While cell viability was not compromised under the experimental conditions, a proinflammatory phenotype was observed in cells incubated with ROFA 100 µg/mL, characterized by increased levels of TNF-α and NO production, together with PM uptake. This inflammatory response seems to precede alterations in redox metabolism, characterized by augmented levels of H2O2, diminished GSH/GSSG ratio, and increased SOD activity. This scenario resulted in increased oxidative damage to phospholipids. Moreover, alterations in mitochondrial respiration were observed following ROFA incubation, such as diminished coupling efficiency and spare respiratory capacity, together with augmented proton leak. These findings were accompanied by a decrease in mitochondrial membrane potential. Finally, NADPH oxidase (NOX) and mitochondria were identified as the main sources of superoxide anion () in our model. These results indicate that PM exposure induces direct activation of macrophages, leading to inflammation and increased reactive oxygen species production through NOX and mitochondria, which impairs antioxidant defense and may cause mitochondrial dysfunction.

Assuntos

Macrófagos Alveolares/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , NADPH Oxidases/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Material Particulado/toxicidade , Superóxidos/metabolismo , Poluentes Atmosféricos/toxicidade , Animais , Antioxidantes/metabolismo , Cinza de Carvão/toxicidade , Peróxido de Hidrogênio/metabolismo , Inflamação , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Camundongos , Mitocôndrias/imunologia , Mitocôndrias/metabolismo , Oxirredução , Estresse Oxidativo/imunologia , Células RAW 264.7 , Fator de Necrose Tumoral alfa/metabolismo

RESUMO

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy specific liver disease characterized by pruritus, elevated serum bile acids and abnormal liver function that may be associated with severe adverse pregnancy outcomes. We previously reported that plasma coenzyme Q10 (CoQ10) is decreased in women with ICP as it is its analogue coenzyme Q9 (CoQ9) in rats with ethinyl estradiol (EE)-induced cholestasis. The aim of the present study was to evaluate the possible therapeutic role of CoQ10 in experimental hepatocellular cholestasis and to compare it with ursodeoxycholic acid (UDCA) supplementation. Bile acids, CoQ9, CoQ10, transaminases, alkaline phosphatase, retinol, α-tocopherol, ascorbic acid, thiobarbituric acid reactive substances, carbonyls, glutathione, superoxide dismutase and catalase were assessed in plasma, liver and/or hepatic mitochondria in control and cholestatic rats supplemented with CoQ10 (250 mg/kg) administered alone or combined with UDCA (25 mg/kg). CoQ10 supplementation prevented bile flow decline (P < 0.05) and the increase in serum alkaline phosphatase and bile acids, particularly lithocholic acid (P < 0.05) in cholestatic rats. Furthermore, it also improved oxidative stress parameters in the liver, increased both CoQ10 and CoQ9 plasma levels and partially prevented the fall in α-tocopherol (P < 0.05). UDCA also prevented cholestasis, but it was less efficient than CoQ10 to improve the liver redox environment. Combined administration of CoQ10 and UDCA resulted in additive effects. In conclusion, present findings show that CoQ10 supplementation attenuated EE-induced cholestasis by promoting a favorable redox environment in the liver, and further suggest that it may represent an alternative therapeutic option for ICP.

Assuntos

Colestase Intra-Hepática/tratamento farmacológico , Suplementos Nutricionais , Complicações na Gravidez/tratamento farmacológico , Ubiquinona/análogos & derivados , Animais , Catalase/metabolismo , Colestase Intra-Hepática/metabolismo , Feminino , Glutationa/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Gravidez , Complicações na Gravidez/metabolismo , Ratos Sprague-Dawley , Superóxido Dismutase/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Ubiquinona/farmacologia , Ubiquinona/uso terapêutico , Ácido Ursodesoxicólico/uso terapêutico

RESUMO

AIM: Intrahepatic cholestasis of pregnancy (ICP) is considered a high-risk condition because it may have serious consequences for the fetus health. ICP is characterized by the accumulation of bile acids in maternal serum which contribute to an imbalance between the production of reactive oxygen species and the antioxidant defenses increasing the oxidative stress experienced by the fetus. Previously, it was reported a significant decrease in plasma coenzyme Q10 (CoQ10) in women with ICP. CoQ10 is a redox substance integrated in the mitochondrial respiratory chain and is recognized as a potent antioxidant playing an intrinsic role against oxidative damage. The objective of the present study was to investigate the levels of CoQ10 in umbilical cord blood during normal pregnancy and in those complicated with ICP, all of them compared to the maternal ones. METHODS: CoQ10 levels and bile acid levels in maternal and umbilical cord blood levels during normal pregnancies (n=23) and in those complicated with ICP (n=13), were investigated. RESULTS: A significant decrease in neonate CoQ10 levels corrected by cholesterol (0.105±0.010 vs. 0.069±0.011, P<0.05, normal pregnancy vs. ICP, respectively), together with an increase of total serum bile acids (2.10±0.02 vs. 7.60±2.30, P<0.05, normal pregnancy vs. ICP, respectively) was observed. CONCLUSIONS: A fetus from an ICP mother is exposed to a greater risk derived from oxidative damage. The recognition of CoQ10 deficiency is important since it could be the starting point for a new and safe intervention strategy which can establish CoQ10 as a promising candidate to prevent the risk of oxidative stress.

Assuntos

Ataxia/sangue , Ácidos e Sais Biliares/sangue , Colestase Intra-Hepática/sangue , Sangue Fetal/química , Doenças Mitocondriais/sangue , Debilidade Muscular/sangue , Complicações na Gravidez/sangue , Ubiquinona/análogos & derivados , Ubiquinona/deficiência , Adulto , Ataxia/diagnóstico , Biomarcadores/sangue , Peso ao Nascer , Colesterol/sangue , Ácido Cólico/sangue , Estudos Transversais , Feminino , Feto/metabolismo , Idade Gestacional , Humanos , Recém-Nascido , Doenças Mitocondriais/diagnóstico , Debilidade Muscular/diagnóstico , Oxirredução , Estresse Oxidativo , Gravidez , Estudos Prospectivos , Espécies Reativas de Oxigênio/metabolismo , Ubiquinona/sangue , Adulto Jovem

RESUMO

The aim of the study was to evaluate the time course of the effects of urban air pollutants on the ocular surface, focusing on the morphological changes, the redox balance, and the inflammatory response of the cornea. 8-week-old mice were exposed to urban or filtered air (UA-group and FA-group, respectively) in exposure chambers for 1, 2, 4, and 12 weeks. After each time, the eyes were enucleated and the corneas were isolated for biochemical analysis. UA-group corneas exhibited a continuous increase in NADPH oxidase-4 levels throughout the exposure time, suggesting an increased production of reactive oxygen species (ROS). After 1 week, an early adaptive response to ROS was observed as an increase in antioxidant enzymes. After 4 weeks, the enzymatic antioxidants were decreased, meanwhile an increase of the glutathione was shown, as a later compensatory antioxidant response. However, redox imbalance took place, evidenced by the increased oxidized proteins, which persisted up to 12 weeks. At this time point, corneal epithelium hyperplasia was also observed. The inflammatory response was modulated by the increase in IL-10 levels after 1 week, which early regulates the release of TNF-α and IL-6. These results suggest that air pollution alters the ocular surface, supported by the observed cellular hyperplasia. The redox imbalance and the inflammatory response modulated by IL-10 play a key role in the response triggered by air pollutants on the cornea. Taking into account this time course study, the ocular surface should also be considered as a relevant target of urban air pollutants.

Assuntos

Poluentes Atmosféricos/toxicidade , Poluição do Ar/efeitos adversos , Epitélio Corneano/patologia , Animais , Brasil , Cidades , Epitélio Corneano/efeitos dos fármacos , Hiperplasia/induzido quimicamente , Hiperplasia/patologia , Interleucina-10/metabolismo , Masculino , Camundongos , NADPH Oxidase 4/metabolismo , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Fatores de Tempo , Testes de Toxicidade Subaguda , Testes de Toxicidade Subcrônica

RESUMO

Monohydroxylated polycyclic aromatic hydrocarbons (OH-PAHs), present in human urine at trace concentrations (viz. from ng L-1 to µg L-1), are considered the main biomarkers of human exposure to PAHs. In this work, we report a simple and high-throughput sample treatment platform to facilitate the biomonitoring of OH-PAHs by making it easier, greener and most cost-effective. This platform is based on the integration of analyte extraction and sample cleanup in a single step by the use of supramolecular solvents with restricted access properties (SUPRAS-RAM). The SUPRAS was spontaneously formed in situ in the urine by the addition of a colloidal suspension of decanoic acid in THF. Metabolites from naphthalene, fluorene, phenanthrene and pyrene were quantitatively extracted (absolute recoveries in the range 91-109%). Polysaccharides and proteins in the urine were excluded from extraction by physical and chemical mechanisms, which allowed the direct analysis of the SUPRAS extract by liquid chromatography tandem mass spectrometry. Absolute matrix effects for OH-PAHs were in the range 92-103%. Method quantification limits for OH-PAHs, without the need for evaporation of the SUPRAS extracts, were in the interval 1.0-6.7 ng L-1. The precision, evaluated in terms of repeatability and reproducibility, varied between 1.1 and 13.8%. The method was successfully applied to the analysis of urine from 16 smoking and non-smoking volunteers. Both analytical and operational features of this method make it suitable to evaluate human exposure to PAHs.

Assuntos

Monitoramento Biológico , Poluentes Ambientais/urina , Hidrocarbonetos Policíclicos Aromáticos/urina , Cromatografia Líquida/métodos , Humanos , Hidrocarbonetos Policíclicos Aromáticos/análise , Reprodutibilidade dos Testes , Solventes/análise , Espectrometria de Massas em Tandem/métodos

RESUMO

Coenzyme Q10 (CoQ10) is a mitochondrial respiratory cofactor and potent endogenous antioxidant. In CoQ10-deficient patients, early treatment with high-oral doses (5-50 mg/kg/day) can limit the progression of renal disease and the onset of neurological manifestations. Crystalline CoQ10 is lipophilic, water-insoluble, and poorly absorbed in the gut. Here, CoQ10 showed low bulk density, another important disadvantage in solid oral formulations. Thus, we propose the use of oleogels to maintain dissolved a high-dose of CoQ10 in medium-chain triglyceride (MCT) oil, using ethylcellulose (EC) for gelling, and a surfactant (sorbitan monostearate -SMS- or lecithin). "True gels" were only obtained with the surfactant presence. Thermoreversible oleogels with 1 g of dissolved CoQ10 per 5 g-disk were successfully developed with proved stability and solubility for 12 months (25.0 °C). SMS was better than lecithin as a surfactant because it allowed lower syneresis, higher CoQ10 retention for 12 months, and notably higher oxidative-stability of the MCT-oil, best immobilized by its true gel network. Plastic deformation without fracture was determined under compression, emulating the soft deformation behavior inside the mouth. SMS-oleogels allowed loading a maximal solubilized CoQ10 dose with maximal stability, and may be easier to swallow by CoQ10-deficient patients who suffer from secondary dysphagia.

Assuntos

Antioxidantes/administração & dosagem , Celulose/análogos & derivados , Tensoativos/química , Ubiquinona/análogos & derivados , Administração Oral , Antioxidantes/química , Celulose/química , Química Farmacêutica/métodos , Relação Dose-Resposta a Droga , Estabilidade de Medicamentos , Hexoses/química , Lecitinas/química , Compostos Orgânicos , Solubilidade , Triglicerídeos/química , Ubiquinona/administração & dosagem , Ubiquinona/química