RESUMO
Leukotrienes play a central pathophysiological role in both paediatric and adult asthma. However, 35% to 78% of asthmatics do not respond to leukotriene inhibitors. In this study we tested the role of the LTA4H regulatory variant rs2660845 and age of asthma onset in response to montelukast in ethnically diverse populations. We identified and genotyped 3,594 asthma patients treated with montelukast (2,514 late-onset and 1,080 early-onset) from seven cohorts (UKBiobank, GoSHARE, BREATHE, Tayside RCT, PAGES, GALA II and SAGE). Individuals under montelukast treatment experiencing at least one exacerbation in a 12-month period were compared against individuals with no exacerbation, using logistic regression for each cohort and meta-analysis. While no significant association was found with European late-onset subjects, a meta-analysis of 523 early-onset individuals from European ancestry demonstrated the odds of experiencing asthma exacerbations by carriers of at least one G allele, despite montelukast treatment, were increased (odds-ratio = 2.92, 95%confidence interval (CI): 1.04-8.18, I2 = 62%, p = 0.0412) compared to those in the AA group. When meta-analysing with other ethnic groups, no significant increased risk of asthma exacerbations was found (OR = 1.60, 95% CI: 0.61-4.19, I2 = 85%, p = 0.342). Our study demonstrates that genetic variation in LTA4H, together with timing of asthma onset, may contribute to variability in montelukast response. European individuals with early-onset (≤18y) carrying at least one copy of rs2660845 have increased odd of exacerbation under montelukast treatment, presumably due to the up-regulation of LTA4H activity. These findings support a precision medicine approach for the treatment of asthma with montelukast.
Assuntos
Acetatos/uso terapêutico , Asma/tratamento farmacológico , Ciclopropanos/uso terapêutico , Epóxido Hidrolases/genética , Farmacogenética , Quinolinas/uso terapêutico , Sulfetos/uso terapêutico , Adolescente , Adulto , Idade de Início , Alelos , Antiasmáticos/uso terapêutico , Asma/fisiopatologia , Criança , Pré-Escolar , Estudos Transversais , Europa (Continente) , Feminino , Genótipo , Hospitalização , Humanos , Antagonistas de Leucotrienos/uso terapêutico , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Razão de Chances , Análise de Regressão , Risco , Adulto JovemRESUMO
OBJECTIVES: Exposure to wood dust may lead to impairment of the lung functions. Microsomal epoxide hydrolase enzyme (EPHX1) was shown to take part in protection against oxidative stress. An alteration in enzyme activity might be associated with its gene polymorphisms. In vitro polymorphisms in exons 3 (His113Tyr) and 4 (Arg139His) lead to reduced activity (slow allele) and increased activity (fast allele). Macrophage inflammatory protein 2 (MIP-2) is produced in rat lung epithelial cells after exposure to fine particles. We aimed to investigate the associations between mEPHX1 polymorphisms (in exon 3 and 4) and lung function in furniture workers and assessment of MIP-2 effect. METHODS: Our study was performed on 70 wood dust exposed male workers and 70 matched normal controls subjects. Ventilatory function tests were measured by spirometer, MIP-2 was performed by ELISA methods and EPHX gene was done by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods for each participant. RESULTS: Significant reduction in forced vital capacity (FVC%) and forced expiratory volume in the first second (FEV1) levels in Tyr-Tyr and Tyr-Hist genotypes of EPHX (exon 3) was observed. Reduced peak expiratory flow (PEF) levels and significant rise in MIP-2 levels were detected in Tyr-Tyr genotype. While high significant reduction in FVC% and FEV1 levels were shown in different genotypes in exon 4. Significant rise was observed in MIP-2 levels in Hist-Hist genotype of exon 4. An increase in duration of exposure showed positive correlation with fall in ventilatory functions. CONCLUSIONS: It was concluded that in Hist139Arg of EPHX gene, fast genotype (Arg-Arg) was associated with impaired ventilatory functions.
Assuntos
Epóxido Hidrolases , Madeira , Epóxido Hidrolases/genética , Genótipo , Humanos , Pulmão , Polimorfismo GenéticoRESUMO
BACKGROUND & AIMS: Alcohol-associated liver disease (ALD) is a significant cause of liver-related morbidity and mortality worldwide and with limited therapies. Soluble epoxide hydrolase (sEH; Ephx2) is a largely cytosolic enzyme that is highly expressed in the liver and is implicated in hepatic function, but its role in ALD is mostly unexplored. METHODS: To decipher the role of hepatic sEH in ALD, we generated mice with liver-specific sEH disruption (Alb-Cre; Ephx2fl/fl). Alb-Cre; Ephx2fl/fl and control (Ephx2fl/fl) mice were subjected to an ethanol challenge using the chronic plus binge model of ALD and hepatic injury, inflammation, and steatosis were evaluated under pair-fed and ethanol-fed states. In addition, we investigated the capacity of pharmacologic inhibition of sEH in the chronic plus binge mouse model. RESULTS: We observed an increase of hepatic sEH in mice upon ethanol consumption, suggesting that dysregulated hepatic sEH expression might be involved in ALD. Alb-Cre; Ephx2fl/fl mice presented efficient deletion of hepatic sEH with corresponding attenuation in sEH activity and alteration in the lipid epoxide/diol ratio. Consistently, hepatic sEH deficiency ameliorated ethanol-induced hepatic injury, inflammation, and steatosis. In addition, targeted metabolomics identified lipid mediators that were impacted significantly by hepatic sEH deficiency. Moreover, hepatic sEH deficiency was associated with a significant attenuation of ethanol-induced hepatic endoplasmic reticulum and oxidative stress. Notably, pharmacologic inhibition of sEH recapitulated the effects of hepatic sEH deficiency and abrogated injury, inflammation, and steatosis caused by ethanol feeding. CONCLUSIONS: These findings elucidated a role for sEH in ALD and validated a pharmacologic inhibitor of this enzyme in a preclinical mouse model as a potential therapeutic approach.
Assuntos
Epóxido Hidrolases/metabolismo , Etanol/toxicidade , Hepatopatias Alcoólicas/etiologia , Fígado/patologia , Compostos de Fenilureia/uso terapêutico , Piperidinas/uso terapêutico , Animais , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Epóxido Hidrolases/antagonistas & inibidores , Epóxido Hidrolases/genética , Etanol/administração & dosagem , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/enzimologia , Fígado/imunologia , Hepatopatias Alcoólicas/tratamento farmacológico , Hepatopatias Alcoólicas/patologia , Camundongos , Camundongos Transgênicos , Compostos de Fenilureia/farmacologia , Piperidinas/farmacologiaRESUMO
The nuclear farnesoid X receptor (FXR) and the enzyme soluble epoxide hydrolase (sEH) are validated molecular targets to treat metabolic disorders such as non-alcoholic steatohepatitis (NASH). Their simultaneous modulation inâ vivo has demonstrated a triad of anti-NASH effects and thus may generate synergistic efficacy. Here we report dual FXR activators/sEH inhibitors derived from the anti-asthma drug Zafirlukast. Systematic structural optimization of the scaffold has produced favorable dual potency on FXR and sEH while depleting the original cysteinyl leukotriene receptor antagonism of the lead drug. The resulting polypharmacological activity profile holds promise in the treatment of liver-related metabolic diseases.
Assuntos
Epóxido Hidrolases/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/agonistas , Compostos de Tosil/química , Sítios de Ligação , Domínio Catalítico , Colesterol 7-alfa-Hidroxilase/genética , Colesterol 7-alfa-Hidroxilase/metabolismo , Avaliação Pré-Clínica de Medicamentos , Epóxido Hidrolases/genética , Epóxido Hidrolases/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Indóis , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Simulação de Acoplamento Molecular , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Fenilcarbamatos , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Relação Estrutura-Atividade , Sulfonamidas , Compostos de Tosil/metabolismo , Compostos de Tosil/farmacologiaRESUMO
Elevated triglyceride-rich lipoproteins (TGRL) in circulation is a risk factor for atherosclerosis. TGRL from subjects consuming a high saturated fat test meal elicited a variable inflammatory response in TNFα-stimulated endothelial cells (EC) that correlated strongly with the polyunsaturated fatty acid (PUFA) content. This study investigates how the relative abundance of oxygenated metabolites of PUFA, oxylipins, is altered in TGRL postprandially, and how these changes promote endothelial inflammation. Human aortic EC were stimulated with TNFα and treated with TGRL, isolated from subjects' plasma at fasting and 3.5 hrs postprandial to a test meal high in saturated fat. Endothelial VCAM-1 surface expression stimulated by TNFα provided a readout for atherogenic inflammation. Concentrations of esterified and non-esterified fatty acids and oxylipins in TGRL were quantified by mass spectrometry. Dyslipidemic subjects produced TGRL that increased endothelial VCAM-1 expression by ≥35%, and exhibited impaired fasting lipogenesis activity and a shift in soluble epoxide hydrolase and lipoxygenase activity. Pro-atherogenic TGRL were enriched in eicosapentaenoic acid metabolites and depleted in esterified C18-PUFA-derived diols. Abundance of these metabolites was strongly predictive of VCAM-1 expression. We conclude the altered metabolism in dyslipidemic subjects produces TGRL with a unique oxylipin signature that promotes a pro-atherogenic endothelial phenotype.
Assuntos
Gorduras na Dieta/administração & dosagem , Dislipidemias/sangue , Epóxido Hidrolases/genética , Ácidos Graxos Insaturados/administração & dosagem , Lipoproteínas/sangue , Oxilipinas/administração & dosagem , Triglicerídeos/sangue , Adulto , Idoso , Estudos de Casos e Controles , Linhagem Celular , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Dislipidemias/genética , Dislipidemias/patologia , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Epóxido Hidrolases/metabolismo , Jejum , Ácidos Graxos Insaturados/sangue , Ácidos Graxos Insaturados/classificação , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação , Lipoxigenase/genética , Lipoxigenase/metabolismo , Masculino , Refeições , Pessoa de Meia-Idade , Oxilipinas/sangue , Oxilipinas/classificação , Período Pós-Prandial , Fator de Necrose Tumoral alfa/farmacologia , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
CVD and associated metabolic diseases are linked to chronic inflammation, which can be modified by diet. The objective of the present study was to determine whether there is a difference in inflammatory markers, blood metabolic and lipid panels and lymphocyte gene expression in response to a high-fat dairy food challenge with or without milk fat globule membrane (MFGM). Participants consumed a dairy product-based meal containing whipping cream (WC) high in saturated fat with or without the addition of MFGM, following a 12 h fasting blood draw. Inflammatory markers including IL-6 and C-reactive protein, lipid and metabolic panels and lymphocyte gene expression fold changes were measured using multiplex assays, clinical laboratory services and TaqMan real-time RT-PCR, respectively. Fold changes in gene expression were determined using the Pfaffl method. Response variables were converted into incremental AUC, tested for differences, and corrected for multiple comparisons. The postprandial insulin response was significantly lower following the meal containing MFGM (P < 0·01). The gene encoding soluble epoxide hydrolase (EPHX2) was shown to be more up-regulated in the absence of MFGM (P = 0·009). Secondary analyses showed that participants with higher baseline cholesterol:HDL-cholesterol ratio (Chol:HDL) had a greater reduction in gene expression of cluster of differentiation 14 (CD14) and lymphotoxin ß receptor (LTBR) with the WC+MFGM meal. The protein and lipid composition of MFGM is thought to be anti-inflammatory. These exploratory analyses suggest that addition of MFGM to a high-saturated fat meal modifies postprandial insulin response and offers a protective role for those individuals with higher baseline Chol:HDL.
Assuntos
Suplementos Nutricionais , Expressão Gênica/efeitos dos fármacos , Glicolipídeos/metabolismo , Glicoproteínas/metabolismo , Secreção de Insulina/efeitos dos fármacos , Refeições , Obesidade/metabolismo , Sobrepeso/metabolismo , Período Pós-Prandial/efeitos dos fármacos , Adolescente , Adulto , Idoso , Biomarcadores/sangue , Proteína C-Reativa/metabolismo , Colesterol/sangue , Citocinas/metabolismo , Laticínios , Dieta , Epóxido Hidrolases/genética , Epóxido Hidrolases/metabolismo , Jejum , Ácidos Graxos , Feminino , Glicolipídeos/farmacologia , Glicoproteínas/farmacologia , Humanos , Insulina/sangue , Interleucina-6/metabolismo , Gotículas Lipídicas , Masculino , Membranas/química , Síndrome Metabólica , Pessoa de Meia-Idade , Adulto JovemRESUMO
NEW FINDINGS: What is the central question of this study? Is there a beneficial effect and what are the mechanisms of acute and multiple hyperbaric oxygenation (HBO2 ) exposures on the outcome of cerebral tissue injury induced by a transient middle cerebral artery occlusion model in diabetic female rats? Are 20-hydroxyeicosatetreanoic acid and epoxyeicosatrienoic acids involved? What is the main finding and its importance? Equal reduction of cortical and total infarct size in rats treated with HBO2 and HET0016 (20-hydroxyeicosatetreanoic acid production inhibitor) and significant mRNA upregulation of epoxyeicosatrienoic acid-producing enzymes (Cyp2J3 and Cyp2C11) in treated groups suggest that HBO2 and HET0016 are highly effective stroke treatments and that cytochrome P450 metabolites are involved in this therapeutic effect. We evaluated the effects of acute and repetitive hyperbaric oxygenation (HBO2 ), 20-hydroxyeicosatetreanoic acid (20-HETE) inhibition by N-hydroxy-N'-(4-butyl-2methylphenyl)-formamidine (HET0016) and their combination on experimental stroke outcomes. Streptozotocin-induced type 1 diabetic Sprague-Dawley female rats (n = 42; n = 7 per group), were subjected to 30 min of transient middle cerebral artery occlusion (t-MCAO)-reperfusion and divided into the following groups: (1) control group, without treatment; and groups exposed to: (2) HBO2 ; (3) multiple HBO2 (HBO2 immediately and second exposure 12 h after t-MCAO); (4) HET0016 pretreatment (1 mg kg-1 , 3 days before t-MCAO) combined with HBO2 after t-MCAO; (5) HET0016 treatment (1 h before, during and for 6 h after t-MCAO); and (6) HET0016 treatment followed by HBO2 after t-MCAO. Messenger RNA expression of CYP2J3, CYP2C11, CYP4A1, endothelial nitric oxide synthase and epoxide hydrolase 2 was determined by real-time qPCR. Cortical infarct size and total infarct size were equally and significantly reduced in HBO2 - and HET0016-treated rats. Combined treatment with HET0016 and HBO2 provided no significant additive effect compared with HET0016 treatment only. Messenger RNA of Cyp2J3 was significantly increased in all study groups, and mRNA of Cyp2C11 was significantly increased in the multiple HBO2 group and the HET0016 treatment followed by HBO2 group, compared with the control group. Expression of endothelial nitric oxide synthase was significantly increased after HBO2 treatments, and expression of epoxide hydrolase 2 was increased in all groups compared with the control group. In diabetic female Sprague-Dawley rats, HBO2 and HET0016 are highly effective stroke treatments, suggesting the involvement of cytochrome P450 metabolites and the NO pathway in this therapeutic effect.
Assuntos
Amidinas/farmacologia , Encéfalo/efeitos dos fármacos , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/terapia , Ácidos Hidroxieicosatetraenoicos/metabolismo , Oxigenoterapia Hiperbárica , Infarto da Artéria Cerebral Média/terapia , Fármacos Neuroprotetores/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Animais , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Terapia Combinada , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Família 2 do Citocromo P450/genética , Família 2 do Citocromo P450/metabolismo , Família 4 do Citocromo P450/genética , Família 4 do Citocromo P450/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/fisiopatologia , Epóxido Hidrolases/genética , Epóxido Hidrolases/metabolismo , Feminino , Regulação Enzimológica da Expressão Gênica , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/fisiopatologia , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/fisiopatologia , Esteroide 16-alfa-Hidroxilase/genética , Esteroide 16-alfa-Hidroxilase/metabolismo , Fatores de TempoRESUMO
N-3 polyunsaturated fatty acids (PUFAs) improve endothelial function. The arachidonic acid-derived metabolites (epoxyeicosatrienoic acids (EETs)) are part of the endothelial hyperpolarization factor and are vasodilators independent of nitric oxide. However, little is known regarding the regulation of EET concentration by docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in blood vessels. Sprague-Dawley rats were fed either a control or fish oil diet for 3 weeks. Compared with the control, the fish oil diet improved acetylcholine-induced vasodilation and reduced the protein expression of soluble epoxide hydrolase (sEH), a key EET metabolic enzyme, in aortic strips. Both DHA and EPA suppressed sEH protein expression in rat aorta endothelial cells (RAECs). Furthermore, the concentration of 4-hydroxy hexenal (4-HHE), a lipid peroxidation product of n-3 PUFAs, increased in n-3 PUFA-treated RAECs. In addition, 4-HHE treatment suppressed sEH expression in RAECs, suggesting that 4-HHE (derived from n-3 PUFAs) is involved in this phenomenon. The suppression of sEH was attenuated by the p38 kinase inhibitor (SB203580) and by treatment with the antioxidant N-acetyl-L-cysteine. In conclusion, sEH expression decreased after n-3 PUFAs treatment, potentially through oxidative stress and p38 kinase. Mild oxidative stress induced by n-3 PUFAs may contribute to their cardio-protective effect.
Assuntos
Ácidos Docosa-Hexaenoicos/farmacologia , Ácido Eicosapentaenoico/farmacologia , Células Endoteliais/efeitos dos fármacos , Epóxido Hidrolases/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Acetilcolina/farmacologia , Ração Animal/análise , Animais , Antígenos CD , Aorta/efeitos dos fármacos , Caderinas , Suplementos Nutricionais , Ácidos Docosa-Hexaenoicos/química , Ácido Eicosapentaenoico/química , Células Endoteliais/metabolismo , Epóxido Hidrolases/genética , Óleos de Peixe/química , Análise de Alimentos , Genes Supressores de Tumor , Proteínas Nucleares , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Artéria Renal/citologia , Vasodilatação/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
BACKGROUND: Neuropathic pain is a debilitating condition with no adequate therapy. The health benefits of omega-3 fatty acids are established, however, the role of docosahexaenoic acid (DHA) in limiting pain has only recently been described and the mechanisms of this action remain unknown. DHA is metabolized into epoxydocosapentanoic acids (EDPs) via cytochrome P450 (CYP450) enzymes which are substrates for the soluble epoxide hydrolase (sEH) enzyme. Here, we tested several hypotheses; first, that the antinociceptive action of DHA is mediated by the EDPs. Second, based on evidence that DHA and CYP450 metabolites elicit analgesia through opioid signalling, we investigated this as a possible mechanism of action. Third, we tested whether the analgesia mediated by epoxy fatty acids had similar rewarding effects as opioid analgesics. METHODS: We tested diabetic neuropathic wild-type and sEH null mice in a conditioned place preference assay for their response to EDPs, sEHI and antagonism of these treatments with naloxone, a mu-opioid receptor antagonist. RESULTS: The EDPs and sEH inhibitors were efficacious against chronic pain, and naloxone antagonized the action of both EDPs and sEH inhibitors. Despite this antagonism, the sEH inhibitors lacked reward side effects differing from opioids. CONCLUSIONS: The EpFA are analgesic against chronic pain differing from opioids which have limited efficacy in chronic conditions. SIGNIFICANCE: EDPs and sEHI mediate analgesia in modelled chronic pain and this analgesia is blocked by naloxone. However, unlike opioids, sEHI are highly effective in neuropathic pain models and importantly lack rewarding side effects.
Assuntos
Analgesia , Analgésicos não Narcóticos/uso terapêutico , Neuropatias Diabéticas/tratamento farmacológico , Ácidos Docosa-Hexaenoicos/uso terapêutico , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Diabetes Mellitus Experimental/complicações , Neuropatias Diabéticas/psicologia , Ácidos Docosa-Hexaenoicos/metabolismo , Epóxido Hidrolases/genética , Epóxido Hidrolases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Naloxona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Receptores Opioides/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
The zinc metallopeptidase insulin regulated aminopeptidase (IRAP), which is highly expressed in the hippocampus and other brain regions associated with cognitive function, has been identified as a high-affinity binding site of the hexapeptide angiotensin IV (Ang IV). This hexapeptide is thought to facilitate learning and memory by binding to the catalytic site of IRAP to inhibit its enzymatic activity. In support of this hypothesis, low molecular weight, nonpeptide specific inhibitors of IRAP have been shown to enhance memory in rodent models. Recently, it was demonstrated that linear and macrocyclic Ang IV-derived peptides can alter the shape and increase the number of dendritic spines in hippocampal cultures, properties associated with enhanced cognitive performance. After screening a library of 10â¯500 drug-like substances for their ability to inhibit IRAP, we identified a series of low molecular weight aryl sulfonamides, which exhibit no structural similarity to Ang IV, as moderately potent IRAP inhibitors. A structural and biological characterization of three of these aryl sulfonamides was performed. Their binding modes to human IRAP were explored by docking calculations combined with molecular dynamics simulations and binding affinity estimations using the linear interaction energy method. Two alternative binding modes emerged from this analysis, both of which correctly rank the ligands according to their experimental binding affinities for this series of compounds. Finally, we show that two of these drug-like IRAP inhibitors can alter dendritic spine morphology and increase spine density in primary cultures of hippocampal neurons.
Assuntos
Cistinil Aminopeptidase/antagonistas & inibidores , Espinhas Dendríticas/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Hipocampo/citologia , Sulfonamidas/farmacologia , Animais , Antígenos CD13/metabolismo , Células Cultivadas , Técnicas de Cocultura , Cistinil Aminopeptidase/metabolismo , Espinhas Dendríticas/enzimologia , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/síntese química , Epóxido Hidrolases/genética , Epóxido Hidrolases/metabolismo , Células HEK293 , Hipocampo/efeitos dos fármacos , Hipocampo/enzimologia , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Ligação Proteica , Ratos Sprague-Dawley , Proteínas Recombinantes/genética , Sulfonamidas/síntese químicaRESUMO
Potato epoxide hydrolase 1 (StEH1) is a biocatalytically important enzyme that exhibits rich enantio- and regioselectivity in the hydrolysis of chiral epoxide substrates. In particular, StEH1 has been demonstrated to enantioconvergently hydrolyze racemic mixes of styrene oxide (SO) to yield (R)-1-phenylethanediol. This work combines computational, crystallographic and biochemical analyses to understand both the origins of the enantioconvergent behavior of the wild-type enzyme, as well as shifts in activities and substrate binding preferences in an engineered StEH1 variant, R-C1B1, which contains four active site substitutions (W106L, L109Y, V141K and I155V). Our calculations are able to reproduce both the enantio- and regioselectivities of StEH1, and demonstrate a clear link between different substrate binding modes and the corresponding selectivity, with the preferred binding modes being shifted between the wild-type enzyme and the R-C1B1 variant. Additionally, we demonstrate that the observed changes in selectivity and the corresponding enantioconvergent behavior are due to a combination of steric and electrostatic effects that modulate both the accessibility of the different carbon atoms to the nucleophilic side chain of D105, as well as the interactions between the substrate and protein amino acid side chains and active site water molecules. Being able to computationally predict such subtle effects for different substrate enantiomers, as well as to understand their origin and how they are affected by mutations, is an important advance towards the computational design of improved biocatalysts for enantioselective synthesis.
Assuntos
Domínio Catalítico , Epóxido Hidrolases/química , Epóxido Hidrolases/metabolismo , Solanum tuberosum/enzimologia , Epóxido Hidrolases/genética , Compostos de Epóxi/química , Compostos de Epóxi/metabolismo , Simulação de Dinâmica Molecular , Mutação , Estereoisomerismo , Especificidade por Substrato , TermodinâmicaRESUMO
The aim of this study was to identify differently expressed proteins in the presence and absence of EPHX2 gene in mouse hypothalamus using proteomics profiling and bioinformatics analysis. This study was performed on 3 wild type (WT) and 3 EPHX2 gene global knockout (KO) mice (EPHX2(-/-)). Using the nano- electrospray ionization (ESI)-LC-MS/MS detector, we identified 31 over-expressed proteins in WT mouse hypothalamus compared to the KO counterparts. Gene Ontology (GO) annotation in terms of the protein-protein interaction network indicated that cellular metabolic process, protein metabolic process, signaling transduction and protein post-translation biological processes involved in EPHX2(-/-) regulatory network. In addition, signaling pathway enrichment analysis also highlighted chronic neurodegenerative diseases and some other signaling pathways, such as TGF-beta signaling pathway, T cell receptor signaling pathway, ErbB signaling pathway, Neurotrophin signaling pathway and MAPK signaling pathway, were strongly coupled with EPHX2 gene knockout. Further studies into the molecular functions of EPHX2 gene in hypothalamus will help to provide new perspective in neurogenesis.
Assuntos
Epóxido Hidrolases/genética , Deleção de Genes , Hipotálamo/fisiologia , Neurogênese/fisiologia , Mapas de Interação de Proteínas/fisiologia , Transdução de Sinais/fisiologia , Animais , Análise por Conglomerados , Biologia Computacional , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Componente Principal , Proteômica , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
OBJECTIVE: Gene × diet interaction plays an important role in atherosclerosis, an inflammatory disorder. Leukotrienes are the most potent inflammatory mediators, and genetic variants encoding leukotriene genes have been implicated in atherosclerosis. This study tests nutrigenetic interaction of a previously defined leukotriene haplotype on carotid artery hypertrophy and atherosclerosis in American Indians. METHODS: This study included 3402 American Indians participating in the Strong Heart Family Study (SHFS). Carotid artery measurements, including intima-media thickness (IMT), vascular mass, and plaque, were assessed using ultrasound. Eleven tagSNPs in the leukotriene A4 hydrolase (LTA4H) gene were genotyped in all subjects. Main haplotype effect and haplotype × diet interaction were examined by generalized estimating equation, adjusting for known risk factors. RESULTS: There was no significant main effect of haplotype or diet on any of the carotid artery measures. However, a previously defined LTA4H haplotype, called HapE, significantly interacted with dietary intake of n-3 and n-6 fatty acids on both IMT (P(HapE × n3) = 0.018, P(HapE × n6) = 0.040) and vascular mass (P(HapE × n3) = 0.012, P(HapE × n6) = 0.018), but not plaque. The direction of this nutrigenetic interaction on IMT was consistent with that reported in a recent study of Caucasian twins. CONCLUSION: Dietary intake of polyunsaturated fatty acids significantly modifies the effect of a leukotriene haplotype on carotid artery hypertrophy but not atherosclerosis in American Indians, independent of established cardiovascular risk factors. Replication of nutrigenetic interaction in two distinct ethnic groups suggests the robustness and generalizability of our findings to diverse populations.
Assuntos
Artérias Carótidas/patologia , Gorduras na Dieta/administração & dosagem , Epóxido Hidrolases/genética , Indígenas Norte-Americanos/genética , Adulto , Artérias Carótidas/diagnóstico por imagem , Doenças das Artérias Carótidas/etiologia , Doenças das Artérias Carótidas/patologia , Espessura Intima-Media Carotídea , Ácidos Graxos Ômega-3/administração & dosagem , Ácidos Graxos Ômega-6/administração & dosagem , Feminino , Genótipo , Haplótipos , Humanos , Hipertrofia/patologia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo ÚnicoRESUMO
OBJECTIVE: Regulation of angiogenesis is critical for many diseases. Specifically, pathological retinal neovascularization, a major cause of blindness, is suppressed with dietary ω3-long-chain polyunsaturated fatty acids (ω3LCPUFAs) through antiangiogenic metabolites of cyclooxygenase and lipoxygenase. Cytochrome P450 epoxygenases (CYP2C8) also metabolize LCPUFAs, producing bioactive epoxides, which are inactivated by soluble epoxide hydrolase (sEH) to transdihydrodiols. The effect of these enzymes and their metabolites on neovascularization is unknown. APPROACH AND RESULTS: The mouse model of oxygen-induced retinopathy was used to investigate retinal neovascularization. We found that CYP2C (localized in wild-type monocytes/macrophages) is upregulated in oxygen-induced retinopathy, whereas sEH is suppressed, resulting in an increased retinal epoxide:diol ratio. With a ω3LCPUFA-enriched diet, retinal neovascularization increases in Tie2-driven human-CYP2C8-overexpressing mice (Tie2-CYP2C8-Tg), associated with increased plasma 19,20-epoxydocosapentaenoic acid and retinal epoxide:diol ratio. 19,20-Epoxydocosapentaenoic acids and the epoxide:diol ratio are decreased with overexpression of sEH (Tie2-sEH-Tg). Overexpression of CYP2C8 or sEH in mice does not change normal retinal vascular development compared with their wild-type littermate controls. The proangiogenic role in retina of CYP2C8 with both ω3LCPUFA and ω6LCPUFA and antiangiogenic role of sEH in ω3LCPUFA metabolism were corroborated in aortic ring assays. CONCLUSIONS: Our results suggest that CYP2C ω3LCPUFA metabolites promote retinal pathological angiogenesis. CYP2C8 is part of a novel lipid metabolic pathway influencing retinal neovascularization.
Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Ácidos Graxos Ômega-3/toxicidade , Macrófagos/enzimologia , Monócitos/enzimologia , Neovascularização Retiniana/induzido quimicamente , Animais , Ácido Araquidônico/metabolismo , Hidrocarboneto de Aril Hidroxilases/genética , Biotransformação , Hipóxia Celular , Citocromo P-450 CYP2C8 , Gorduras na Dieta/farmacocinética , Ácidos Docosa-Hexaenoicos/metabolismo , Ácido Eicosapentaenoico/metabolismo , Epóxido Hidrolases/deficiência , Epóxido Hidrolases/genética , Epóxido Hidrolases/fisiologia , Proteínas do Olho/metabolismo , Ácidos Graxos Ômega-3/administração & dosagem , Ácidos Graxos Ômega-3/classificação , Ácidos Graxos Ômega-3/farmacocinética , Ácidos Graxos Insaturados/administração & dosagem , Ácidos Graxos Insaturados/farmacocinética , Humanos , Lipoxigenase/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oxigênio/toxicidade , Prostaglandina-Endoperóxido Sintases/metabolismo , RNA Mensageiro/biossíntese , Receptor TIE-2/genética , Proteínas Recombinantes de Fusão/metabolismo , Neovascularização Retiniana/prevenção & controleRESUMO
AIM: To investigate the relationship between cerebroprotection of pinocembrin and epoxyeicosatrienoic acids (EETs) and their regulating enzyme soluble epoxide hydrolase (sEH). METHODS: Rats underwent middle cerebral artery occlusion (MCAO) to mimic permanent focal ischemia, and pinocembrin was administrated via tail vein injection at 10 min, 4 h, 8 h and 23 h after MCAO. After 24 MCAO, rats were re-anesthetized, and the blood and brain were harvested and analyzed. RESULTS: Pinocembrin displayed significant protective effects on MCAO rats indicated by reduced neurological deficits and infarct volume. Importantly, co-administration of 0.2 mg·kg(-1) 14, 15-EEZE, a putative selective EET antagonist, weakened the beneficial effects of pinocembrin. 14, 15-EET levels in the blood and brain of rats after 24 h MCAO were elevated in the presence of pinocembrin. In an assay for hydrolase activity, pinocembrin significantly lowered brain sEH activity of MCAO rats and inhibited recombinant human sEH activity in a concentration-dependent manner (IC50, 2.58 µmol·L(-1)). In addition, Western blot and immunohistochemistry analysis showed that pinocembrin at doses of 10 mg·kg(-1) and 30 mg·kg(-1) significantly down-regulated sEH protein in rat brain, especially the hippocampus CA1 region of MCAO rats. CONCLUSION: Inhibiting sEH and then increasing the potency of EETs may be one of the mechanisms through which pinocembrin provides cerebral protection.
Assuntos
Ácidos Araquidônicos/metabolismo , Isquemia Encefálica/enzimologia , Isquemia Encefálica/prevenção & controle , Epóxido Hidrolases/metabolismo , Flavanonas/administração & dosagem , Substâncias Protetoras/administração & dosagem , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Encéfalo/metabolismo , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/genética , Isquemia Encefálica/metabolismo , Modelos Animais de Doenças , Epóxido Hidrolases/genética , Humanos , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
There are several studies supporting the role of HMG-CoA reductase inhibitors such as atorvastatin against carcinogenesis, in which inhibiting the generation of prenyl intermediates involved in protein prenylation plays the crucial role. Mutation of Kras gene is the most common genetic alteration in pancreatic cancer and the Ras protein requires prenylation for its membrane localization and activity. In the present study, the effectiveness of atorvastatin against pancreatic carcinogenesis and its effect on protein prenylation were determined using the LSL-KrasG12D-LSL-Trp53R172H-Pdx1-Cre mouse model (called Pankras/p53 mice). Five-week-old Pankras/p53 mice were fed either an AIN93M diet or a diet supplemented with 100 ppm atorvastatin. Kaplan-Meier survival analysis with Log-Rank test revealed a significant increase in survival in mice fed 100 ppm atorvastatin (171.9 ± 6.2 d) compared to the control mice (144.9 ± 8.4 d, P < 0.05). Histologic and immunohistochemical analysis showed that atorvastatin treatment resulted in a significant reduction in tumor volume and Ki-67-labeled cell proliferation. Mechanistic studies on primary pancreatic tumors and the cultured murine pancreatic carcinoma cells revealed that atorvastatin inhibited prenylation in several key proteins, including Kras protein and its activities, and similar effect was observed in pancreatic carcinoma cells treated with farnesyltransferase inhibitor R115777. Microarray assay on the global gene expression profile demonstrated that a total of 132 genes were significantly modulated by atorvastatin; and Waf1p21, cyp51A1, and soluble epoxide hydrolase were crucial atorvastatin-targeted genes which involve in inflammation and carcinogenesis. This study indicates that atorvastatin has the potential to serve as a chemopreventive agent against pancreatic carcinogenesis.
Assuntos
Carcinoma Ductal Pancreático/prevenção & controle , Transformação Celular Neoplásica/efeitos dos fármacos , Ácidos Heptanoicos/farmacologia , Proteínas de Homeodomínio/genética , Neoplasias Pancreáticas/prevenção & controle , Proteínas Proto-Oncogênicas p21(ras)/genética , Pirróis/farmacologia , Transativadores/genética , Proteína Supressora de Tumor p53/genética , Animais , Atorvastatina , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Epóxido Hidrolases/genética , Epóxido Hidrolases/metabolismo , Proteínas de Homeodomínio/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Camundongos , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Pâncreas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fosforilação/efeitos dos fármacos , Prenilação/efeitos dos fármacos , Prenilação/genética , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transativadores/metabolismo , Transcriptoma/efeitos dos fármacosRESUMO
The EPXH2 gene encodes soluble epoxide hydrolase (sEH), which has two distinct enzyme activities: epoxide hydrolase (Cterm-EH) and phosphatase (Nterm-phos). The Cterm-EH is involved in the metabolism of arachidonic acid epoxides that play important roles in blood pressure, cell growth, inflammation, and pain. While recent findings suggested complementary biological roles for Nterm-phos, research is limited by the lack of potent bioavailable inhibitors of this phosphatase activity. Also, a potent bioavailable inhibitor of this activity could be important in the development of therapy for cardiovascular diseases. We report herein the development of an HTS enzyme-based assay for Nterm-phos (Z'>0.9) using AttoPhos as the substrate. This assay was used to screen a wide variety of chemical entities, including a library of known drugs that have reached through clinical evaluation (Pharmakon 1600), as well as a library of pesticides and environmental toxins. We discovered that ebselen inhibits sEH phosphatase activity. Ebselen binds to the N-terminal domain of sEH (K(I)=550 nM) and chemically reacts with the enzyme to quickly and irreversibly inhibit Nterm-phos, and subsequently Cterm-EH, and thus represents a new class of sEH inhibitor.
Assuntos
Epóxido Hidrolases/análise , Ensaios de Triagem em Larga Escala , Bibliotecas de Moléculas Pequenas/química , Azóis/metabolismo , Epóxido Hidrolases/genética , Epóxido Hidrolases/metabolismo , Humanos , Isoindóis , Cinética , Compostos Organosselênicos/metabolismo , Praguicidas/metabolismo , Ligação Proteica , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Especificidade por Substrato , Toxinas Biológicas/metabolismoRESUMO
The EPXH2 gene encodes for the soluble epoxide hydrolase (sEH), which has two distinct enzyme activities: epoxide hydrolase (Cterm-EH) and phosphatase (Nterm-phos). The Cterm-EH is involved in the metabolism of epoxides from arachidonic acid and other unsaturated fatty acids, endogenous chemical mediators that play important roles in blood pressure regulation, cell growth, inflammation and pain. While recent findings suggested complementary biological roles for Nterm-phos, its mode of action is not well understood. Herein, we demonstrate that lysophosphatidic acids are excellent substrates for Nterm-phos. We also showed that sEH phosphatase activity represents a significant (20-60%) part of LPA cellular hydrolysis, especially in the cytosol. This possible role of sEH on LPA hydrolysis could explain some of the biology previously associated with the Nterm-phos. These findings also underline possible cellular mechanisms by which both activities of sEH (EH and phosphatase) may have complementary or opposite roles.
Assuntos
Epóxido Hidrolases/metabolismo , Lisofosfolipídeos/metabolismo , Epóxido Hidrolases/química , Epóxido Hidrolases/genética , Humanos , Organofosfatos/química , Organofosfatos/isolamento & purificação , Organofosfatos/metabolismo , Bibliotecas de Moléculas Pequenas , Solubilidade , Especificidade por SubstratoRESUMO
The aim of our present study was to examine the regulation of xenobiotic- and antioxidant enzymes by phytogenic feed additives in the intestine and the liver of broilers. A total of 240 male Ross-308 broiler chickens (1 d old) were fed a commercial starter diet for 2 weeks. On day 15, the birds were assigned to six treatment groups of forty birds each. The control (Con) group was fed a diet without any additive for 3 weeks. The diet of group sulforaphane (SFN) contained broccoli extract providing 0.075 g/kg SFN, whereas the diets of the other four groups contained 0.15 g/kg essential oils from turmeric (Cuo), oregano (Oo), thyme and rosemary (Ro). Weight gain and feed conversion were slightly impaired by Cuo and Oo. In the jejunum SFN, Cuo and Ro increased the expression of xenobiotic enzymes (epoxide hydrolases 1 and 2 and aflatoxin B1 aldehyde reductase) and of the antioxidant enzyme haeme oxygenase regulated by an 'antioxidant response element' (ARE) compared to group Con. In contrast to our expectations in the liver, the expression of these enzymes was decreased by all the additives. Nevertheless, all the additives increased the Trolox equivalent antioxidant capacity of the jejunum and the liver and reduced Fe-induced lipid peroxidation in the liver. We conclude that the up-regulation of ARE genes in the small intestine reduces oxidative stress in the organism and represents a novel mechanism by which phytogenic feed additives improve the health of farm animals.
Assuntos
Ração Animal/análise , Antioxidantes/administração & dosagem , Brassica/química , Galinhas/crescimento & desenvolvimento , Indução Enzimática , Óleos Voláteis/administração & dosagem , Extratos Vegetais/administração & dosagem , Aldeído Redutase/genética , Aldeído Redutase/metabolismo , Animais , Antioxidantes/metabolismo , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Galinhas/metabolismo , Colo/enzimologia , Colo/crescimento & desenvolvimento , Colo/metabolismo , Curcuma/química , Epóxido Hidrolases/genética , Epóxido Hidrolases/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Mucosa Intestinal/enzimologia , Mucosa Intestinal/crescimento & desenvolvimento , Mucosa Intestinal/metabolismo , Jejuno/enzimologia , Jejuno/crescimento & desenvolvimento , Jejuno/metabolismo , Lamiaceae/química , Fígado/enzimologia , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Masculino , Brotos de Planta/química , RNA Mensageiro/metabolismo , Aumento de PesoRESUMO
In cruciferous vegetables, myrosinase metabolizes the relatively inactive glucosinolates into isothiocyanates and other products that have the ability to increase detoxification enzyme expression. Thus, maintaining myrosinase activity during food preparation may be critical to receiving the maximum benefit of consumption of Brussels sprouts or other cruciferous vegetables. To test the importance of maintaining myrosinase activity for maximizing bioactivity, experimental diets containing 20% unblanched (active myrosinase) or 20% blanched (inactivated myrosinase) freeze-dried Brussels sprouts and a nutrient-matched control diet were evaluated in vitro and in vivo for their ability to induce detoxification enzymes. Treatment of immortalized HepG2 human hepatoma cells with the unblanched Brussels sprout diet caused a greater increase quinone activity compared to the blanched Brussels sprout diet. C3H/HeJ mice fed the unblanched Brussels sprout diets for 2 wk had significantly higher plasma sulforaphane concentrations. Liver expression of CYP1A1 and epoxide hydrolase, measured using real-time PCR, was correlated with the plasma concentration of sulforaphane. In the lung, expression of epoxide hydrolase, thioredoxin reductase, UDP glucuronosyltransferase, quinone reductase, heme oxygenase, CYP1A1, CYP1A2, and CYP1B1 were also correlated with the plasma concentration of sulforaphane. Together these data demonstrate that, as predicted by the in vitro experiment, in vivo exposure to Brussels sprouts with active myrosinase resulted in greater induction of both phase I and phase II detoxification enzymes in the liver and the lungs that correlated with plasma sulforaphane concentrations.