RESUMO
The widespread presence of 3-monochloropropane-1,2-diol (3-MCPD) and glycidol in refined edible oils have raised food industrial and public health concerns, but their specific biomarkers of exposure and urinary metabolic pathways indicating nephrotoxicity remain largely unknown. Here, we unraveled the in vivo biotransformation of these two contaminants and revealed how they affect metabolic pathways in rats. Urine metabolomes in rats administered with glycidol or 3-MCPD were investigated using ultra-high performance liquid chromatography combined with a quadrupole-orbitrap high-resolution mass spectrometry. Compared to the currently acknowledged metabolite which is only 2,3-dihydroxypropyl mercapturic acid, we identified 8 and 4 new specific exposure biomarkers of glycidol and 3-MCPD, respectively, via mapping the glyceryl polymerization and glutathione and sulfur conjugation. The changes of metabolites in the surrounding metabolic network were investigated to further gain insight into their metabolic fates. Exposure to glycidol up-regulated citrate, isocitrate, ketoglutarate, malate, and pyruvate in the tricarboxylic acid cycle and glycolysis pathways, while 3-MCPD intake down-regulated these signal molecules in both pathways. Nonetheless, L-cysteine, proline, and arginine were significantly decreased by the effect of either glycidol or 3-MCPD. Our findings first map the urinary metabolomics of both contaminants from edible oils and advance the omics-level recognition for their observational health hazards.
Assuntos
alfa-Cloridrina , Acetilcisteína/análogos & derivados , Animais , Compostos de Epóxi , Óleos de Plantas/química , Propanóis , Ratos , Toxicocinética , alfa-Cloridrina/análise , alfa-Cloridrina/toxicidadeRESUMO
The objective of this work was to study the effects and mechanisms of S-allylmercapto-N-acetylcysteine (ASSNAC) in the treatment of pulmonary emphysema based on network pharmacology analysis and other techniques. Firstly, the potential targets associated with ASSNAC and COPD were integrated using public databases. Then, a protein-protein interaction network was constructed using String database and Cytoscape software. The Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes pathway analysis were performed on DAVID platform. The molecular docking of ASSNAC with some key disease targets was implemented on the SwissDock platform. To verify the results of the network pharmacology, a pulmonary emphysema mice model was established and treated with ASSNAC. Besides, the expressions of the predicted targets were detected by immunohistochemistry, Western blot analysis or enzyme-linked immunosorbent assay. Results showed that 33 overlapping targets are achieved, including CXCL8, ICAM1, MAP2K1, PTGS2, ACE and so on. The critical pathways of ASSNAC against COPD involved arachidonic acid metabolism, chemokine pathway, MAPK pathway, renin-angiotensin system, and others. Pharmacodynamic experiments demonstrated that ASSNAC decreased the pulmonary emphysema and inflammation in the pulmonary emphysema mice. Therefore, these results confirm the perspective of network pharmacology in the target verification, and indicate the treatment potential of ASSNAC against COPD.
Assuntos
Acetilcisteína/análogos & derivados , Compostos Alílicos/farmacologia , Anti-Inflamatórios/farmacologia , Enfisema Pulmonar/tratamento farmacológico , Acetilcisteína/farmacologia , Acetilcisteína/uso terapêutico , Compostos Alílicos/uso terapêutico , Animais , Anti-Inflamatórios/uso terapêutico , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Quimiocinas/metabolismo , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Humanos , Masculino , Camundongos , Simulação de Acoplamento Molecular , Farmacologia em Rede , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas/efeitos dos fármacos , Mapas de Interação de Proteínas/imunologia , Enfisema Pulmonar/diagnóstico , Enfisema Pulmonar/imunologia , Enfisema Pulmonar/patologia , Sistema Renina-Angiotensina/efeitos dos fármacos , Sistema Renina-Angiotensina/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologiaRESUMO
Fennel and other herbs contain the secondary plant metabolites estragole and trans-anethole, of which estragole is carcinogenic in rodents. It is metabolically activated by cytochrome P450-catalyzed conversion to 1'-hydroxyestragole and subsequent sulfo conjugation to the genotoxic 1'-sulfoxyestragole. The current study followed the hypothesis that the reactive sulfate ester may be detoxified by glutathione conjugation, leading to the urinary excretion of a resultant mercapturic acid. We identified the assumed downstream metabolite N-acetyl-S-[3'-(4-methoxyphenyl)allyl]-l-Cys (AMPAC) in human urine samples after consumption of fennel tea. An isotope-dilution technique for its quantification by ultraperformance liquid chromatography-tandem mass spectrometry and [13C3,15N]AMPAC in urine samples was developed. The method was applied to determine the AMPAC concentration in urine samples following uptake of 500 mL of fennel tea containing 2.2 mg of estragole by 12 healthy participants (six females and six males). Before drinking the tea, the urinary AMPAC concentration was below the limit of detection. In most of the participants, the highest amounts of urinary AMPAC were found in the first-hour urine after exposure. The excretion by first-order kinetics (range of t1/2 = 0.78-1.54 h; mean ± SD: 1.13 ± 0.21 h) led to a nearly complete clearance within 8 h in all participants. The total AMPAC excreted was in the range of 93-1076 ng, reflecting pronounced interindividual variations of enzymes taking part in estragole metabolism. Importantly, AMPAC was also formed in one volunteer following oral uptake of a single dose of isolated trans-anethole, albeit to a much smaller extent compared to estragole. AMPAC may be of future use as a human biomarker for the internal exposure to the carbocation formed from either 1'-sulfoxyestragole or 3'-sulfoxyisoestragole, the reactive sulfate ester metabolites of estragole and trans-anethole, respectively.
Assuntos
Acetilcisteína/análogos & derivados , Acetilcisteína/urina , Anisóis/farmacocinética , Foeniculum , Chá , Adulto , Derivados de Alilbenzenos , Feminino , Frutas , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
The exchange proteins activated by cAMP (Epacs) have been shown to play important roles in producing inflammation-induced nociception. Transient receptor potential vanilloid type 1 (TRPV1) is a major receptor processing thermal and chemosensitive nociceptive information. The role of Epacs in modulating the activity of TRPV1 has yet to be determined. Studying the effect of complete Freund adjuvant (CFA)-induced inflammation on capsaicin-activated TRPV1 nociceptive responses in dorsal root ganglia (DRG), we found that CFA produced a large increase in capsaicin-induced responses. The increase was inhibited by Epac1 and Epac2 antagonists. Thus, activation of Epacs is critical in producing enhancement in TRPV1-mediated responses under inflammatory conditions. In addition, the inflammation-induced enhancement of TRPV1 responses was blocked by PKCα and PKCε inhibitors, suggesting the essential roles of these PKCs in enhancing TRPV1 responses. To determine the mechanism underlying the Epac actions on TRPV1, we studied the effects of the Epac activator, 8-(4-chlorophenylthio)-2-O-methyl-cAMP (CPT), on capsaicin-induced nociceptive behavioral responses, capsaicin-activated currents, expression and membrane trafficking of PKC and TRPV1 in DRG. CPT was found to enhance capsaicin-induced nociception and ionic currents. The enhancement was inhibited by PKCα and PKCε inhibitors. In addition, CPT increased the expression of phosphorylated PKCα (pPKCα) and membrane TRPV1 expression in DRG. Studying the colocalization of TRPV1 and pPKCα or pPKCε in DRG slices prepared from CFA-treated rats, we found that pPKCα or pPKCε expressed with TRPV1 in different-sized neurons to exert differential influences on TRPV1 activity. Thus, Epac-PKC signaling is critically important in producing inflammation-induced potentiation of TRPV1 functions.
Assuntos
Acetilcisteína/análogos & derivados , Eritromicina/análogos & derivados , Hiperalgesia/fisiopatologia , Inflamação/enzimologia , Proteína Quinase C-épsilon/metabolismo , Transdução de Sinais/fisiologia , Canais de Cátion TRPV/metabolismo , Acetilcisteína/metabolismo , Acetilcisteína/farmacologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Biotinilação , Capsaicina/toxicidade , Células Cultivadas , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Eritromicina/metabolismo , Eritromicina/farmacologia , Adjuvante de Freund/toxicidade , Gânglios Espinais/citologia , Hiperalgesia/patologia , Inflamação/induzido quimicamente , Masculino , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Técnicas de Patch-Clamp , Proteína Quinase C-alfa/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2X4/metabolismoRESUMO
Alzheimer's disease is a progressive neurodegenerative disease for which there is no cure and only a few treatments providing little relief. Increased oxidative stress that is associated with aging is strongly implicated in the pathogenesis and progression of Alzheimer's disease. Studies have shown that levels of the endogenous antioxidant glutathione decline at an early stage of Alzheimer's disease with decreased levels correlating with worse cognitive functions. N-acetylcysteine, a drug also widely available as a dietary supplement, is a precursor of L-cysteine, which in turn is a component of glutathione. Because cysteine availability is a limiting factor for glutathione synthesis, treatment with N-acetylcysteine may increase glutathione levels and thereby counter oxidative stress, promote redox -regulated cell signaling, and improve immune responses. In this review, we evaluate the existing literature and the potential of N-acetylcysteine in promoting cognitive health and alleviating cognitive decline associated with dementia. Discussion will also include possible mechanisms of action of N-acetylcysteine, its effects on aging biology, and safety of long-term use. Based on the available literature, a nutraceutical formulation containing N-acetylcysteine among other compounds has shown some pro-cognitive benefits in Alzheimer's patients and older adults, but the evidence for N-acetylcysteine alone is less robust. Although N-acetylcysteine crosses the blood-brain-barrier, low bioavailability is an obstacle. One promising avenue of research may be to explore derivatives of N-acetylcysteine such as N-acetylcysteine amide, which has been reported in preclinical studies to have higher permeability through cellular and mitochondrial membranes with increased central nervous system bioavailability compared to N-acetylcysteine.
Assuntos
Acetilcisteína/uso terapêutico , Envelhecimento Cognitivo , Demência/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacocinética , Humanos , Fármacos Neuroprotetores/farmacocinética , Nootrópicos/farmacocinética , Nootrópicos/uso terapêuticoRESUMO
Garlic has shown versatile medicinal activities in the prevention and treatment of diseases such as chronic obstructive pulmonary disease (COPD). However, no individual garlic bioactive components have yet been determined in the COPD treatment effects. In this work, S-allylmercapto-l-cysteine (SAMC) identified in the aged garlic was selected as a model compound to determine its COPD therapeutic potential. The COPD model was established by using lipopolysaccharides (LPS) to stimulate the human airway submucosal gland cell line SPC-A1. Previous studies show that both MUC5AC up-regulation and AQP5 down-regulation play an important role in viscous COPD mucus secretions. The modulation effects of SAMC on LPS-induced MUC5AC and AQP5 productions in SPC-A1 cells were then evaluated. Pretreatment of the SPC-A1 cells with SAMC attenuated MUC5AC secretion and increased AQP5 expression in a dose-dependent manner in the non-cytotoxic concentration range of 20 to 100µM. Mechanistic studies suggested that SAMC could suppress the accumulation of MUC5AC mRNA and inhibit IкBα degradation and NF-кB p65 translocation. These results suggest that SAMC could be a promising candidate in the prevention and treatment of MUC5AC-associated disorders such as COPD.
Assuntos
Acetilcisteína/análogos & derivados , Compostos Alílicos/uso terapêutico , Aquaporina 5/metabolismo , Células Epiteliais/efeitos dos fármacos , Alho/imunologia , Mucina-5AC/metabolismo , NF-kappa B/metabolismo , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Acetilcisteína/uso terapêutico , Aquaporina 5/genética , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Células Epiteliais/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Lipopolissacarídeos/imunologia , Mucina-5AC/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
An 8-week feeding trial was conducted to evaluate the effect of N-acetyl cysteine (NAC) and glycine supplementation on growth performance, glutathione (GSH) synthesis, anti-oxidative and immune ability of Nile tilapia, Oreochromis niloticus. Four practical diets were formulated, control, control +0.2% NAC, control +0.5% glycine, control +0.2% NAC +0.5% glycine. Each diet was randomly assigned to quadruplicate groups of 30 fish (approximately 9.5 g). The weight gain and specific growth rate were significantly increased with the supplementation of NAC and glycine. While they had no effect on feed efficiency feed intake and survival. Glutathion peroxidase (GPx) was increased by NAC and γ-glutamine cysteine synthase (γ-GCS) in plasma were increased by glycine. After the feeding trail, fish were challenged by Streptococcus iniae, fish fed the diet supplemented with NAC obtained significantly higher survival rate after 72 h challenge test. NAC also decreased malonaldehyde (MDA) in liver, increased glutathione S-transferase (GST) activity in plasma, up-regulated mRNA expression of Superoxide dismutase (SOD) and GPx in liver and headkidney. Dietary supplementation of glycine increased the anti-oxidative ability of tilapia through increase anti-oxidative enzyme activity (SOD, glutathione reductase, myeloperoxidase) and up-regulate anti-oxidative gene expression (SOD). Immune ability only enhanced by the supplementation of NAC through increased interleukin-1ß (IL-1ß) mRNA expression. These results clearly indicated that the supplementation of NAC and glycine can significantly improve the growth performance of tilapia, and NAC also enhance the anti-oxidative and immune capacity of tilapia, glycine could only enhance the anti-oxidative ability.
Assuntos
Acetilcisteína/metabolismo , Ciclídeos , Doenças dos Peixes/imunologia , Glicina/metabolismo , Imunidade Inata , Infecções Estreptocócicas/veterinária , Acetilcisteína/análogos & derivados , Acetilcisteína/análise , Ração Animal/análise , Animais , Antioxidantes/metabolismo , Ciclídeos/crescimento & desenvolvimento , Ciclídeos/metabolismo , Dieta/veterinária , Suplementos Nutricionais/análise , Doenças dos Peixes/microbiologia , Glutationa/metabolismo , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/microbiologia , Streptococcus iniae/fisiologiaRESUMO
Proteasomes are intracellular complexes that control selective protein degradation in organisms ranging from Archaea to higher eukaryotes. These structures have multiple proteolytic activities that are required for cell differentiation, replication and maintaining cellular homeostasis. Here, we document the presence of the 20S proteasome in the protist parasite Tritrichomonas foetus. Complementary techniques, such as a combination of whole genome sequencing technologies, bioinformatics algorithms, cell fractionation and biochemistry and microscopy approaches were used to characterise the 20S proteasome of T. foetus. The 14 homologues of the typical eukaryotic proteasome subunits were identified in the T. foetus genome. Alignment analyses showed that the main regulatory and catalytic domains of the proteasome were conserved in the predicted amino acid sequences from T. foetus-proteasome subunits. Immunofluorescence assays using an anti-proteasome antibody revealed a labelling distributed throughout the cytosol as punctate cytoplasmic structures and in the perinuclear region. Electron microscopy of a T. foetus-proteasome-enriched fraction confirmed the presence of particles that resembled the typical eukaryotic 20S proteasome. Fluorogenic assays using specific peptidyl substrates detected presence of the three typical peptidase activities of eukaryotic proteasomes in T. foetus. As expected, these peptidase activities were inhibited by lactacystin, a well-known specific proteasome inhibitor, and were not affected by inhibitors of serine or cysteine proteases. During the transformation of T. foetus to endoflagellar form (EFF), also known as pseudocyst, we observed correlations between the EFF formation rates, increases in the proteasome activities and reduced levels of ubiquitin-protein conjugates. The growth, cell cycle and EFF transformation of T. foetus were inhibited after treatment with lactacystin in a dose-dependent manner. Lactacystin treatment also resulted in an accumulation of ubiquitinated proteins and caused increase in the amount of endoplasmic reticulum membranes in the parasite. Taken together, our results suggest that the ubiquitin-proteasome pathway is required for cell cycle and EFF transformation in T. foetus.
Assuntos
Ciclo Celular , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Protozoários/metabolismo , Esporos de Protozoários/metabolismo , Tritrichomonas foetus/metabolismo , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacologia , Sequência de Aminoácidos , Western Blotting , Inibidores de Cisteína Proteinase/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Flagelos/metabolismo , Flagelos/ultraestrutura , Estágios do Ciclo de Vida/efeitos dos fármacos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Dados de Sequência Molecular , Filogenia , Complexo de Endopeptidases do Proteassoma/classificação , Complexo de Endopeptidases do Proteassoma/genética , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/ultraestrutura , Homologia de Sequência de Aminoácidos , Esporos de Protozoários/efeitos dos fármacos , Esporos de Protozoários/ultraestrutura , Tritrichomonas foetus/genética , Tritrichomonas foetus/crescimento & desenvolvimentoRESUMO
Thrombocytopenia in methotrexate (MTX)-treated cancer and rheumatoid arthritis (RA) patients connotes the interference of MTX with platelets. Hence, it seemed appealing to appraise the effect of MTX on platelets. Thereby, the mechanism of action of MTX on platelets was dissected. MTX (10 µM) induced activation of pro-apoptotic proteins Bid, Bax and Bad through JNK phosphorylation leading to ΔΨm dissipation, cytochrome c release and caspase activation, culminating in apoptosis. The use of specific inhibitor for JNK abrogates the MTX-induced activation of pro-apoptotic proteins and downstream events confirming JNK phosphorylation by MTX as a key event. We also demonstrate that platelet mitochondria as prime sources of ROS which plays a central role in MTX-induced apoptosis. Further, MTX induces oxidative stress by altering the levels of ROS and glutathione cycle. In parallel, the clinically approved thiol antioxidant N-acetylcysteine (NAC) and its derivative N-acetylcysteine amide (NACA) proficiently alleviate MTX-induced platelet apoptosis and oxidative damage. These findings underpin the dearth of research on interference of therapeutic drugs with platelets, despite their importance in human health and disease. Therefore, the use of antioxidants as supplementary therapy seems to be a safe bet in pathologies associated with altered platelet functions.
Assuntos
Acetilcisteína/análogos & derivados , Acetilcisteína/farmacologia , Antimetabólitos Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , MAP Quinase Quinase 4/genética , Metotrexato/farmacologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Regulação da Expressão Gênica , Humanos , MAP Quinase Quinase 4/metabolismo , Potencial da Membrana Mitocondrial , Metotrexato/antagonistas & inibidores , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Oxirredução , Estresse Oxidativo , Fosforilação , Cultura Primária de Células , Transdução de Sinais , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína de Morte Celular Associada a bcl/genética , Proteína de Morte Celular Associada a bcl/metabolismoRESUMO
Aberrations in the ubiquitin-proteasome system (UPS) are implicated in the pathogenesis of various diseases. Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamines biosynthesis, is involved in hypertension development. In this study we investigated whether UPS regulated TH turnover in PC12 cells and hypothalamic and brainstem neurons from spontaneously hypertensive rats (SHR) and whether this system was impaired in hypertension. PC12 cells were exposed to proteasome or lysosome inhibitors and TH protein level evaluated by Western blot. Lactacystin, a proteasome inhibitor, induced an increase of 86 ± 15% in TH levels after 30 min of incubation, then it started to decrease up to 6 h to reach control levels and finally it rose up to 35.2 ± 8.5% after 24 h. Bafilomycin, a lysosome inhibitor, did not alter TH protein levels during short times, but it increased TH by 92 ± 22% above basal after 6 h treatment. Before degradation proteasome substrates are labeled by conjugation with ubiquitin. Efficacy of proteasome inhibition on TH turnover was evidenced by accumulation of ubiquitinylated TH after 30 min. Further, the inhibition of proteasome increased the quantity of TH phosphorylated at Ser40, which is essential for TH activity, by 2.7 ± 0.3 fold above basal. TH protein level was upregulated in neurons from hypothalami and brainstem of SHR when the proteasome was inhibited during 30 min, supporting that neuronal TH is also short-term regulated by the proteasome. Since the increased TH levels reported in hypertension may result from proteasome dysfunction, we evaluate proteasome activity. Proteasome activity was significantly reduced by 67 ± 4% in hypothalamic and brainstem neurons from SHR while its protein levels did not change. Present findings show that TH is regulated by the UPS. The impairment in proteasome activity observed in SHR neurons may be one of the causes of the increased TH protein levels reported in hypertension.
Assuntos
Tronco Encefálico/metabolismo , Hipertensão/metabolismo , Hipotálamo/metabolismo , Neurônios/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Ubiquitina/metabolismo , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacologia , Animais , Tronco Encefálico/citologia , Hipotálamo/citologia , Masculino , Células PC12 , Inibidores de Proteassoma/farmacologia , Ratos , Ratos Endogâmicos SHR , Ratos Wistar , Tirosina 3-Mono-Oxigenase/genéticaRESUMO
Lysosomal storage disorders (LSD) are a group of heterogeneous diseases caused by compromised enzyme function leading to multiple organ failure. Therapeutic approaches involve enzyme replacement (ERT), which is effective for a substantial fraction of patients. However, there are still concerns about a number of issues including tissue penetrance, generation of host antibodies against the therapeutic enzyme, and financial aspects, which render this therapy suboptimal for many cases. Treatment with pharmacological chaperones (PC) was recognized as a possible alternative to ERT, because a great number of mutations do not completely abolish enzyme function, but rather trigger degradation in the endoplasmic reticulum. The theory behind PC is that they can stabilize enzymes with remaining function, avoid degradation and thereby ameliorate disease symptoms. We tested several compounds in order to identify novel small molecules that prevent premature degradation of the mutant lysosomal enzymes α-galactosidase A (for Fabry disease (FD)) and acid α-glucosidase (GAA) (for Pompe disease (PD)). We discovered that the expectorant Ambroxol when used in conjunction with known PC resulted in a significant enhancement of mutant α-galactosidase A and GAA activities. Rosiglitazone was effective on α-galactosidase A either as a monotherapy or when administered in combination with the PC 1-deoxygalactonojirimycin. We therefore propose both drugs as potential enhancers of pharmacological chaperones in FD and PD to improve current treatment strategies.
Assuntos
1-Desoxinojirimicina/análogos & derivados , Ambroxol/farmacologia , Ativadores de Enzimas/farmacologia , Lisossomos/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , alfa-Galactosidase/genética , alfa-Glucosidases/genética , 1-Desoxinojirimicina/farmacologia , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacologia , Bezafibrato/farmacologia , Doença de Fabry/tratamento farmacológico , Doença de Fabry/enzimologia , Expressão Gênica , Doença de Depósito de Glicogênio Tipo II/tratamento farmacológico , Doença de Depósito de Glicogênio Tipo II/enzimologia , Células HEK293 , Humanos , Leupeptinas/farmacologia , Lisossomos/metabolismo , Pioglitazona , Plasmídeos/química , Plasmídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Estabilidade Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tiazolidinedionas/farmacologia , Transfecção , alfa-Galactosidase/metabolismo , alfa-Glucosidases/metabolismoRESUMO
This study investigated the effect of polymorphic genes GSTT1, GSTM1, GSTA1, EHPX1, NQO1, CYP2E1, CYP1A and MPO on the urinary concentrations and ratio (R) of the benzene metabolites trans,trans-muconic acid (t,t-MA) and S-phenyl mercapturic acid (S-PMA) in 301 oil refinery workers. The metabolites' concentrations are lower and R is higher (100.66) in non-smokers (n=184) than in smokers (n=117, R=36.54). Non-smokers have lower S-PMA and a higher R in GSTT1 null genotypes than in positive, and a higher S-PMA and a lower R in GSTA1 wild type genotypes. In smokers the GSTT1 null genotype effect on both S-PMA and R is confirmed, and is also shown in GSTM1 null, but not in GSTA1 wild type genotypes. GSTT1 null polymorphism reduces the conjugation rate of benzene epoxide with GSH, and to a lesser extent also GSTTA1 mutant, GSTM1 null and NQO1 mutant genotypes. The activity of one GST is compensated by another in GSTM1 and GSTA1 defective subjects, but not in GSTT1 null genotypes, whose average S-PMA excretion is about 50% with respect to the positive ones, for the same benzene exposure. R showed to be a more sensitive marker for these effects than the metabolite levels.
Assuntos
Acetilcisteína/análogos & derivados , Benzeno/metabolismo , Polimorfismo Genético , Fumar/efeitos adversos , Ácido Sórbico/análogos & derivados , Acetilcisteína/metabolismo , Adulto , Feminino , Regulação da Expressão Gênica , Genótipo , Humanos , Indústrias , Masculino , Exposição Ocupacional , Petróleo , Ácido Sórbico/metabolismoRESUMO
Mitochondrial dysfunction is becoming a pivotal target for neuroprotective strategies following contusion spinal cord injury (SCI) and the pharmacological compounds that maintain mitochondrial function confer neuroprotection and improve long-term hindlimb function after injury. In the current study we evaluated the efficacy of cell-permeating thiol, N-acetylcysteine amide (NACA), a precursor of endogenous antioxidant glutathione (GSH), on mitochondrial function acutely, and long-term tissue sparing and hindlimb locomotor recovery following upper lumbar contusion SCI. Some designated injured adult female Sprague-Dawley rats (n=120) received either vehicle or NACA (75, 150, 300 or 600mg/kg) at 15min and 6h post-injury. After 24h the total, synaptic, and non-synaptic mitochondrial populations were isolated from a single 1.5cm spinal cord segment (centered at injury site) and assessed for mitochondrial bioenergetics. Results showed compromised total mitochondrial bioenergetics following acute SCI that was significantly improved with NACA treatment in a dose-dependent manner, with maximum effects at 300mg/kg (n=4/group). For synaptic and non-synaptic mitochondria, only 300mg/kg NACA dosage showed efficacy. Similar dosage (300mg/kg) also maintained mitochondrial GSH near normal levels. Other designated injured rats (n=21) received continuous NACA (150 or 300mg/kg/day) treatment starting at 15min post-injury for one week to assess long-term functional recovery over 6weeks post-injury. Locomotor testing and novel gait analyses showed significantly improved hindlimb function with NACA that were associated with increased tissue sparing at the injury site. Overall, NACA treatment significantly maintained acute mitochondrial bioenergetics and normalized GSH levels following SCI, and prolonged delivery resulted in significant tissue sparing and improved recovery of hindlimb function.
Assuntos
Acetilcisteína/análogos & derivados , Metabolismo Energético/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Recuperação de Função Fisiológica/efeitos dos fármacos , Traumatismos da Medula Espinal/tratamento farmacológico , Acetilcisteína/uso terapêutico , Animais , Modelos Animais de Doenças , Método Duplo-Cego , Sistemas de Liberação de Medicamentos , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/patologia , Coxeadura Animal/tratamento farmacológico , Coxeadura Animal/etiologia , Mitocôndrias/enzimologia , Atividade Motora/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Medula Espinal/patologia , Medula Espinal/ultraestrutura , Traumatismos da Medula Espinal/complicações , Sinapses/efeitos dos fármacos , Sinapses/enzimologia , Sinapses/patologia , Sinapses/ultraestrutura , Fatores de TempoRESUMO
Traumatic brain injury (TBI) has become a growing epidemic but no approved pharmacological treatment has been identified. Our previous work indicates that mitochondrial oxidative stress/damage and loss of bioenergetics play a pivotal role in neuronal cell death and behavioral outcome following experimental TBI. One tactic that has had some experimental success is to target glutathione using its precursor N-acetylcysteine (NAC). However, this approach has been hindered by the low CNS bioavailability of NAC. The current study evaluated a novel, cell permeant amide form of N-acetylcysteine (NACA), which has high permeability through cellular and mitochondrial membranes resulting in increased CNS bioavailability. Cortical tissue sparing, cognitive function and oxidative stress markers were assessed in rats treated with NACA, NAC, or vehicle following a TBI. At 15days post-injury, animals treated with NACA demonstrated significant improvements in cognitive function and cortical tissue sparing compared to NAC or vehicle treated animals. NACA treatment also was shown to reduce oxidative damage (HNE levels) at 7days post-injury. Mechanistically, post-injury NACA administration was demonstrated to maintain levels of mitochondrial glutathione and mitochondrial bioenergetics comparable to sham animals. Collectively these data provide a basic platform to consider NACA as a novel therapeutic agent for treatment of TBI.
Assuntos
Acetilcisteína/análogos & derivados , Lesões Encefálicas/complicações , Lesões Encefálicas/tratamento farmacológico , Metabolismo Energético/efeitos dos fármacos , Aprendizagem em Labirinto/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Acetilcisteína/uso terapêutico , Aldeídos/metabolismo , Animais , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/patologia , Córtex Cerebral/ultraestrutura , Modelos Animais de Doenças , Método Duplo-Cego , Glutationa/metabolismo , Masculino , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
CONTEXT: Glutaredoxins (GRX) are involved in the regulation of thiol redox state. GRX-1 is a cytosolic enzyme responsible for the catalysis of deglutathionylation of proteins. To date, very few inhibitors of GRX-1 have been reported. OBJECTIVE: The objective of this paper is to report 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethyl-sulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA) as an inhibitor of human GRX-1. MATERIALS AND METHODS: The mechanism of inhibition of GRX-1 was investigated using dialysis, substrate protection, and mass spectrometry. RESULTS: 2-AAPA inhibits GRX-1 in a time and concentration dependent manner. The activity did not return following dialysis indicating that inhibition is irreversible. Results of substrate protection and mass spectrometry indicate that the inhibition is occurring at the active site. The compound also produced GRX inhibition in human ovarian cancer cells. DISCUSSION: 2-AAPA is an irreversible GRX-1 inhibitor with similar or greater potency compared to previously reported inhibitors. CONCLUSION: The inhibition of GRX-1 by 2-AAPA could be used as a tool to study thiol redox state.
Assuntos
Acetilcisteína/análogos & derivados , Inibidores Enzimáticos/farmacologia , Glutarredoxinas/antagonistas & inibidores , Tiocarbamatos/farmacologia , Acetilcisteína/farmacologia , Linhagem Celular Tumoral/efeitos dos fármacos , Diálise , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Glutationa/metabolismo , Humanos , Tiocarbamatos/químicaRESUMO
A study of workers exposed to jet fuel propellant 8 (JP-8) was conducted at U.S. Air Force bases and included the evaluation of three biomarkers of exposure: S-benzylmercapturic acid (BMA), S-phenylmercapturic acid (PMA), and (2-methoxyethoxy)acetic acid (MEAA). Postshift urine specimens were collected from various personnel categorized as high (n = 98), moderate (n = 38) and low (n = 61) JP-8 exposure based on work activities. BMA and PMA urinary levels were determined by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), and MEAA urinary levels were determined by gas chromatography-mass spectrometry (GC-MS). The numbers of samples determined as positive for the presence of the BMA biomarker (above the test method's limit of detection [LOD = 0.5 ng/ml]) were 96 (98.0%), 37 (97.4%), and 58 (95.1%) for the high, moderate, and low (control) exposure workgroup categories, respectively. The numbers of samples determined as positive for the presence of the PMA biomarker (LOD = 0.5 ng/ml) were 33 (33.7%), 9 (23.7%), and 12 (19.7%) for the high, moderate, and low exposure categories. The numbers of samples determined as positive for the presence of the MEAA biomarker (LOD = 0.1 µ g/ml) were 92 (93.4%), 13 (34.2%), and 2 (3.3%) for the high, moderate, and low exposure categories. Statistical analysis of the mean levels of the analytes demonstrated MEAA to be the most accurate or appropriate biomarker for JP-8 exposure using urinary concentrations either adjusted or not adjusted for creatinine; mean levels of BMA and PMA were not statistically significant between workgroup categories after adjusting for creatinine.
Assuntos
Acetatos/urina , Hidrocarbonetos/farmacocinética , Militares , Exposição Ocupacional , Petróleo/metabolismo , Urinálise/métodos , Acetilcisteína/análogos & derivados , Acetilcisteína/urina , Adulto , Aeroportos , Biomarcadores/urina , Cromatografia Líquida de Alta Pressão , Creatinina/urina , Relação Dose-Resposta a Droga , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Hidrocarbonetos/administração & dosagem , Limite de Detecção , Instalações Militares , Espectrometria de Massas em Tandem , Estados UnidosRESUMO
Oxidative stress (OS) resulting from an imbalance between antioxidant defenses and the intracellular accumulation of reactive oxygen species (ROS) contributes to age-related memory deficits. While impaired synaptic plasticity in neuronal networks is thought to underlie cognitive deficits during aging, whether this process is targeted by OS and what the mechanisms involved are still remain open questions. In this study, we investigated the age-related effects of the reducing agent N-acetyl-L-cysteine (L-NAC) on the activation of the N-methyl-D-aspartate receptor (NMDA-R) by its co-agonist D-serine, because alterations in this mechanism contribute greatly to synaptic plasticity deficits in aged animals. Long-term dietary supplementation with L-NAC prevented oxidative damage in the hippocampus of aged rats. Electrophysiological recordings in the CA1 of hippocampal slices indicated that NMDA-R-mediated synaptic potentials and theta-burst-induced long-term potentiation (LTP) were depressed in aged animals, deficits that could be reversed by exogenous D-serine. Chronic treatment with L-NAC, but not acute application of the reducing agent, restored potent D-serine-dependent NMDA-R activation and LTP induction in aged rats. In addition, it is also revealed that the age-related decrease in D-serine levels and in the expression of the synthesizing enzyme serine racemase, which underlies the decrease in NMDA-R activation by the amino acid, was rescued by long-term dietary treatment with L-NAC. Our results indicate that protecting redox status in aged animals could prevent injury to the cellular mechanisms underlying cognitive aging, in part by maintaining potent NMDA-R activation through the D-serine-dependent pathway.
Assuntos
Envelhecimento , Hipocampo/metabolismo , Plasticidade Neuronal , Estresse Oxidativo , Receptores de N-Metil-D-Aspartato/metabolismo , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacologia , Animais , Lisina/análogos & derivados , Lisina/farmacologia , Masculino , Plasticidade Neuronal/efeitos dos fármacos , Ratos , Ratos Wistar , Serina/metabolismoRESUMO
OBJECTIVE: To study the effect of epigallocatechin gallate (EGCG) against lactacystin induced PC12 cell injury. METHODS: The inoculated rat PC12 cells were cultured for 24 h, followed by intervention. The cells were divided into 5 groups, i.e., the normal control group, 10 micromol/L lactacystin injury group, and the EGCG pretreated groups (at the final concentration of 5, 10, and 50 micromol/L, respectively). The cytoactive was detected by MTT colorimetry. Morphological changes of the cell nucleus were observed by Hoechst 33,258 staining, and the apoptosis ratio was detected by flow cytometry (FCM). RESULTS: EGCG at different doses showed protective effect on lactacystin-induced PC12 cell injury. Compared with the lactacystin injury group [(61.22 +/- 1.02)%], the cytoactive in EGCG pretreated groups at the final concentration of 5, 10, and 50 micromol/L, respectively increased obviously to (66.99 +/- 1.30)%, (66.67 +/- 0.65)%, and (73.4 +/- 0.67)%, respectively. Hoechst 33 258 staining found that more nuclear pyknosis and aggregation occurred in the lactacystin injury group, but less occurred in EGCG pretreated groups. FCM indicated that the apoptosis ratio was reduced by EGCG pretreatment. It was 3.0%, 60.4%, 59.8%, 57.5%, and 38.6%, respectively in the normal control group, the lactacystin injury group, and EGCG pretreated groups (at the final concentration of 5, 10, and 50 micromol/L, respectively). CONCLUSION: EGCG could attenuate lactacystin induced PC 12 cell injury.
Assuntos
Acetilcisteína/análogos & derivados , Apoptose/efeitos dos fármacos , Catequina/análogos & derivados , Acetilcisteína/efeitos adversos , Animais , Catequina/farmacologia , Citometria de Fluxo , Células PC12 , RatosRESUMO
Impairment of the ubiquitin-proteasome system, responsible for clearing of misfolded and unwanted proteins, has been implicated in the loss of nigrostriatal dopaminergic neurons characteristic of Parkinson's disease (PD). Recently, proteasome inhibitors have been used to model parkinsonian-like changes in animals. In the present study, the effects of intrastriatal and intranigral injections of the selective proteasome inhibitor lactacystin on key markers of PD were examined in Wistar rats. Comparisons of these two different routes of lactacystin administration revealed that only a unilateral, intranigral injection of lactacystin at a dose of 0.5, 1, 2.5 and 5 µg/2 µl produced after 7 days distinct decreases in the concentrations of dopamine (DA) and its metabolites (DOPAC, 3-MT, HVA) in the ipsilateral striatum. The used doses of lactacystin (except for 0.5 µg/2 µl) significantly accelerated DA catabolism, i.e. the total, oxidative MAO-dependent and COMT-catalyzed pathways, as assessed by HVA/DA, DOPAC/DA and 3-MT/DA ratios, respectively, in the ipsilateral striatum. Such alterations were not observed in the striatal DA content and catabolism either 7, 14 or 21 days after a unilateral, intrastriatal high-dose lactacystin injection (5 and 10 µg/2 µl). Intranigrally administered lactacystin (1 µg/2 µl) caused a marked decline of tyrosine hydroxylase (TH) and α-synuclein protein levels in that structure. Neither TH nor α-synuclein protein levels in the substantia nigra (SN) were affected by high lactacystin doses injected intrastriatally. Moreover, stereological counting of TH-immunoreactive neurons and autoradiographic analysis of [(3)H]GBR 12,935 binding to dopamine transporter confirmed a loss of nigrostriatal dopaminergic neurons after an intranigral lactacystin (1 and 2.5 µg/2 µl) injection. An appearance of cardinal neurochemical and histological changes of parkinsonian type only after intranigral lactacystin injection indicates that DA cell bodies in the SN, but not DA terminals in the striatum are susceptible to proteasome inhibition.
Assuntos
Acetilcisteína/análogos & derivados , Corpo Estriado/efeitos dos fármacos , Doença de Parkinson/metabolismo , Inibidores de Proteassoma/farmacologia , Substância Negra/efeitos dos fármacos , Acetilcisteína/administração & dosagem , Acetilcisteína/farmacologia , Animais , Western Blotting , Relação Dose-Resposta a Droga , Masculino , Inibidores de Proteassoma/administração & dosagem , Ratos , Ratos WistarRESUMO
In this work, the metabolite profiles of Danggui Buxue Tang (DBT) in rat bile and plasma were qualitatively described, and the possible metabolic pathways of DBT were subsequently proposed. Emphasis was put on correlative analysis of metabolite profiling in different biological fluids. After oral administration of DBT, bile and plasma samples were collected and pretreated by solid phase extraction. Rapid resolution liquid chromatography coupled to time-of-flight mass spectrometry (RRLC-TOFMS) was used for characterization of DBT-related compounds (parent compounds and metabolites) in biological matrices. A total of 142 metabolites were detected and tentatively identified from the drug-containing bile and plasma samples. Metabolite profiling shows that rat bile contained relatively more glutathione-derived conjugates, more saponins compounds and more diverse forms of metabolites than urine. The metabolite profile in plasma revealed that glucuronide conjugates of isoflavonoids, dimmers, acetylcysteine conjugates and parent form of phthalides, as well as saponin aglycones were the major circulating forms of DBT. Collectively, the metabolite profile analysis of DBT in different biological matrices provided a comprehensive understanding of the in vivo metabolic fates of constituents in DBT.