Assuntos
Antiasmáticos/uso terapêutico , Asma/tratamento farmacológico , Asma/genética , Beclometasona/uso terapêutico , Citocromo P-450 CYP3A/genética , Polimorfismo de Nucleotídeo Único , Administração por Inalação , Adolescente , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/imunologia , Asma/imunologia , Asma/patologia , Criança , Pré-Escolar , Citocromo P-450 CYP3A/imunologia , Gerenciamento Clínico , Feminino , Expressão Gênica , Humanos , Masculino , Resultado do TratamentoRESUMO
Paroxetine is a selective serotonin reuptake inhibitor (SSRI) that is currently available on the market and is suspected of causing congenital malformations in babies born to mothers who take the drug during the first trimester of pregnancy. We utilized organismal performance assays (OPAs), a novel toxicity assessment method, to assess the safety of paroxetine during pregnancy in a rodent model. OPAs utilize genetically diverse wild mice (Mus musculus) to evaluate competitive performance between experimental and control animals as they compete among each other for limited resources in semi-natural enclosures. Performance measures included reproductive success, male competitive ability and survivorship. Paroxetine-exposed males weighed 13% less, had 44% fewer offspring, dominated 53% fewer territories and experienced a 2.5-fold increased trend in mortality, when compared with controls. Paroxetine-exposed females had 65% fewer offspring early in the study, but rebounded at later time points, presumably, because they were no longer exposed to paroxetine. In cages, paroxetine-exposed breeders took 2.3 times longer to produce their first litter and pups of both sexes experienced reduced weight when compared with controls. Low-dose paroxetine-induced health declines detected in this study that were undetected in preclinical trials with doses 2.5-8 times higher than human therapeutic doses. These data indicate that OPAs detect phenotypic adversity and provide unique information that could be useful towards safety testing during pharmaceutical development.
Assuntos
Anormalidades Induzidas por Medicamentos/etiologia , Antidepressivos de Segunda Geração/farmacologia , Peso Corporal/efeitos dos fármacos , Comportamento Competitivo/efeitos dos fármacos , Paroxetina/farmacologia , Reprodução/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Feminino , Masculino , Camundongos , Modelos de Riscos ProporcionaisRESUMO
Inhaled glucocorticoids, such as beclomethasone dipropionate (BDP), are the mainstay treatment of asthma. However, ≈ 30% of patients exhibit little to no benefit from treatment. It has been postulated that glucocorticoid resistance, or insensitivity, is attributable to individual differences in glucocorticoid receptor-mediated processes. It is possible that variations in cytochrome P450 3A enzyme-mediated metabolism of BDP may contribute to this phenomenon. This hypothesis was explored by evaluating the contributions of CYP3A4, 3A5, 3A7, and esterase enzymes in the metabolism of BDP in vitro and relating metabolism to changes in CYP3A enzyme mRNA expression via the glucocorticoid receptor in lung and liver cells. CYP3A4 and CYP3A5 metabolized BDP via hydroxylation ([M4] and [M6]) and dehydrogenation ([M5]) at similar rates; CYP3A7 did not metabolize BDP. A new metabolite [M6], formed by the combined action of esterases and CYP3A4 hydroxylation, was also characterized. To validate the results observed using microsomes and recombinant enzymes, studies were also conducted using A549 lung and DPX2 liver cells. Both liver and lung cells produced esterase-dependent metabolites [M1-M3], with [M1] correlating with CYP3A5 mRNA induction in A549 cells. Liver cells produced both hydroxylated and dehydrogenated metabolites [M4, M5, and M6], but lung cells produced only the dehydrogenated metabolite [M5]. These studies show that CYP3A4 and CYP3A5 metabolize BDP to inactive metabolites and suggest that differences in the expression or function of these enzymes in the lung and/or liver could influence BDP disposition in humans.
Assuntos
Anti-Inflamatórios/metabolismo , Beclometasona/metabolismo , Citocromo P-450 CYP3A/metabolismo , Administração por Inalação , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Primers do DNA , Meia-Vida , Humanos , Isoenzimas/metabolismo , Pulmão/citologia , Pulmão/enzimologia , Pulmão/metabolismo , Reação em Cadeia da Polimerase , Espectrometria de Massas em TandemRESUMO
OBJECTIVE: To determine the relationship between allelic variations in genes involved in fluticasone propionate (FP) metabolism and asthma control among children with asthma managed with inhaled FP. STUDY DESIGN: The relationship between variability in asthma control scores and genetic variation in drug metabolism was assessed by genotyping 9 single nucleotide polymorphisms in the CYP3A4, CYP3A5, and CYP3A7 genes. Genotype information was compared with asthma control scores (0=well controlled to 15=poorly controlled), determined using a questionnaire modified from the National Heart Lung and Blood Institute's Expert Panel 3 guidelines. RESULTS: Our study cohort comprised 734 children with asthma (mean age, 8.8±4.3 years) and was predominantly male (61%) and non-Hispanic white (53%). More than one-half of the children (56%; n=413) were receiving an inhaled glucocorticoid daily, with FP the most frequently prescribed agent (65%). Among the children receiving daily FP, single nucleotide polymorphisms in CYP3A5 and CYP3A7 were not associated with asthma control scores. In contrast, asthma control scores were significantly improved in the 20 children (7%) with the CYP3A4*22 allele (median, 3; range, 0-6) compared with the 201 children without the CYP3A4*22 allele (median, 4; range, 0-15; P=.02). The presence of CYP3A4*22 was associated with improved asthma control scores by 2.1 points (95% CI, 0.5-3.8). CONCLUSION: The presence of CYP3A4*22, which is associated with decreased hepatic CYP3A4 expression and activity, was accompanied by improved asthma control in the FP-treated children. Decreased CYP3A4 activity may improve asthma control with inhaled FP.
Assuntos
Androstadienos/farmacocinética , Asma/tratamento farmacológico , Broncodilatadores/farmacocinética , Citocromo P-450 CYP3A/genética , Administração por Inalação , Adolescente , Androstadienos/administração & dosagem , Broncodilatadores/administração & dosagem , Criança , Pré-Escolar , Feminino , Fluticasona , Humanos , Masculino , Farmacogenética , Polimorfismo Genético , Estudos Prospectivos , Inquéritos e QuestionáriosRESUMO
Inhaled glucocorticoids are the first-line treatment for patients with persistent asthma. However, approximately thirty percent of patients exhibit glucocorticoid insensitivity, which may involve excess metabolic clearance of the glucocorticoids by CYP3A enzymes in the lung. CYP3A4, 3A5, and 3A7 enzymes metabolize glucocorticoids, which in turn induce CYP3A genes. However, the mechanism of CYP3A5 mRNA regulation by glucocorticoids in lung cells has not been determined. In hepatocytes, glucocorticoids bind to the glucocorticoid receptor (GR), which induces the expression of the constitutive androstane receptor or pregnane X receptor; both of which bind to the retinoid X receptor alpha, leading to the induction of CYP3A4, 3A5, and 3A7. There is also evidence to suggest a direct induction of CYP3A5 by GR activation in liver cells. In this study, these pathways were evaluated as the mechanism for CYP3A5 mRNA induction by glucocorticoids in freshly isolated primary tracheal epithelial, adenocarcinomic human alveolar basal epithelial (A549), immortalized bronchial epithelial (BEAS-2B), primary normal human bronchial/tracheal epithelial (NHBE), primary small airway epithelial (SAEC), and primary lobar epithelial lung cells. In A549 cells, beclomethasone 17-monopropionate ([M1]) induced CYP3A5 mRNA through the glucocorticoid receptor. CYP3A5 mRNA induction by five different glucocorticoids was attenuated by inhibiting the glucocorticoid receptor using ketoconazole, and for beclomethasone dipropionate, using siRNA-mediated knock-down of the glucocorticoid receptor. The constitutive androstane receptor was not expressed in lung cells. SAEC cells, a primary lung cell line, expressed CYP3A5, but CYP3A5 mRNA was not induced by glucocorticoid treatment despite evaluating a multitude of cell culture conditions. None of the other lung cells expressed CYP3A4, 3A5 or 3A7 mRNA. These studies demonstrate that CYP3A5 mRNA is induced by glucocorticoids in A549 cells via the glucocorticoid receptor, but that additional undefined regulatory processes exist in primary lung cells.
RESUMO
Asthma is one of the most prevalent diseases in the world, for which the mainstay treatment has been inhaled glucocorticoids (GCs). Despite the widespread use of these drugs, approximately 30% of asthma sufferers exhibit some degree of steroid insensitivity or are refractory to inhaled GCs. One hypothesis to explain this phenomenon is interpatient variability in the clearance of these compounds. The objective of this research is to determine how metabolism of GCs by the CYP3A family of enzymes could affect their effectiveness in asthmatic patients. In this work, the metabolism of four frequently prescribed inhaled GCs, triamcinolone acetonide, flunisolide, budesonide, and fluticasone propionate, by the CYP3A family of enzymes was studied to identify differences in their rates of clearance and to identify their metabolites. Both interenzyme and interdrug variability in rates of metabolism and metabolic fate were observed. CYP3A4 was the most efficient metabolic catalyst for all the compounds, and CYP3A7 had the slowest rates. CYP3A5, which is particularly relevant to GC metabolism in the lungs, was also shown to efficiently metabolize triamcinolone acetonide, budesonide, and fluticasone propionate. In contrast, flunisolide was only metabolized via CYP3A4, with no significant turnover by CYP3A5 or CYP3A7. Common metabolites included 6ß-hydroxylation and Δ(6)-dehydrogenation for triamcinolone acetonide, budesonide, and flunisolide. The structure of Δ(6)-flunisolide was unambiguously established by NMR analysis. Metabolism also occurred on the D-ring substituents, including the 21-carboxy metabolites for triamcinolone acetonide and flunisolide. The novel metabolite 21-nortriamcinolone acetonide was also identified by liquid chromatography-mass spectrometry and NMR analysis.
Assuntos
Antiasmáticos/administração & dosagem , Antiasmáticos/metabolismo , Citocromo P-450 CYP3A/metabolismo , Glucocorticoides/administração & dosagem , Glucocorticoides/metabolismo , Pulmão/enzimologia , Administração por Inalação , Androstadienos/administração & dosagem , Androstadienos/metabolismo , Antiasmáticos/química , Hidrocarboneto de Aril Hidroxilases/metabolismo , Biotransformação , Budesonida/administração & dosagem , Budesonida/metabolismo , Catálise , Cromatografia Líquida de Alta Pressão , Fluocinolona Acetonida/administração & dosagem , Fluocinolona Acetonida/análogos & derivados , Fluocinolona Acetonida/metabolismo , Fluticasona , Glucocorticoides/química , Humanos , Hidroxilação , Isoenzimas , Cinética , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Estrutura Molecular , Proteínas Recombinantes/metabolismo , Triancinolona Acetonida/administração & dosagem , Triancinolona Acetonida/metabolismoRESUMO
This study characterized electrophilic and radical products derived from the metabolism of capsaicin by cytochrome P450 and peroxidase enzymes. Multiple glutathione and ß-mercaptoethanol conjugates (a.k.a., adducts), derived from the trapping of quinone methide and quinone intermediates of capsaicin, its analogue nonivamide, and O-demethylated and aromatic hydroxylated metabolites thereof, were produced by human liver microsomes and individual recombinant human P450 enzymes. Conjugates derived from concomitant dehydrogenation of the alkyl terminus of capsaicin were also characterized. Modifications to the 4-OH substituent of the vanilloid ring of capsaicinoids largely prevented the formation of electrophilic intermediates, consistent with the proposed structures and mechanisms of formation for the various conjugates. 5,5'-Dicapsaicin, presumably arising from the bimolecular coupling of free radical intermediates was also characterized. Finally, the analysis of hepatic glutathione conjugates and urinary N-acetylcysteine conjugates from mice dosed with capsaicin confirmed the formation of glutathione conjugates of O-demethylated quinone methide and 5-OH-capsaicin in vivo. These data demonstrated that capsaicin and structurally similar analogues are converted to reactive intermediates by certain P450 enzymes, which may partially explain conflicting reports related to the cytotoxic, pro-carcinogenic, and chemoprotective effects of capsaicinoids in different cells and/or organ systems.
Assuntos
Capsaicina/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Acetilcisteína/química , Acetilcisteína/metabolismo , Acetilcisteína/urina , Animais , Capsaicina/análogos & derivados , Capsaicina/análise , Cromatografia Líquida de Alta Pressão , Sistema Enzimático do Citocromo P-450/genética , Dimerização , Glutationa/química , Glutationa/metabolismo , Humanos , Indolquinonas/análise , Indolquinonas/metabolismo , Fígado/química , Fígado/metabolismo , Camundongos , Isótopos de Oxigênio/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Massas em TandemRESUMO
Cytochrome P450 2F1 (P450 2F1) is expressed exclusively in the human respiratory tract and is implicated in 3-methylindole (3MI)-induced pneumotoxicity via dehydrogenation of 3MI to a reactive electrophilic intermediate, 3-methyleneindolenine (3-MEI). Studies of P450 2F1 to date have been limited by the failure to express this enzyme in Escherichia coli. By contrast, P450 2F3, a caprine homologue that shares 84% sequence identity with P450 2F1 (86 amino acid differences), has been expressed in E. coli at yields greater than 250 nmol/L culture. We hypothesized that a limited number of sequence differences between P450s 2F1 and 2F3 could limit P450 2F1 expression in E. coli and that problematic P450 2F1 sequence elements could be identified by directed evolution. A library of P450 2F1/2F3 mutants was created by DNA family shuffling and screened for expression in E. coli. Three generations of DNA shuffling revealed a mutant (named JH_2F_F3_1_007) with 96.5% nucleotide sequence identity to P450 2F1 and which expressed 119 ± 40 pmol (n = 3, mean ± SD) hemoprotein in 1 mL microaerobic cultures. Across all three generations, two regions were observed where P450 2F3-derived sequence was consistently substituted for P450 2F1 sequence in expressing mutants, encoding nine amino acid differences between P450s 2F1 and 2F3: nucleotides 191-278 (amino acids 65-92) and 794-924 (amino acids 265-305). Chimeras constructed to specifically test the importance of these two regions confirmed that P450 2F3 sequence is essential in both regions for expression in E. coli but that other non-P450 2F1 sequence elements outside of these regions also improved the expression of mutant JH_2F_F3_1_007. Mutant JH_2F_F3_1_007 catalyzed the dehydrogenation of 3MI to 3-MEI as indicated by the observation of glutathione adducts after incubation in the presence of glutathione. The JH_2F_F3_1_007 protein differs from P450 2F1 at only 20 amino acids and should facilitate further studies of the structure-activity relationships of P450s of the 2F subfamily.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Evolução Molecular Direcionada , Escherichia coli/metabolismo , Cromatografia Líquida de Alta Pressão , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Glutationa/metabolismo , Humanos , Indóis/química , Espectrometria de Massas , Modelos Moleculares , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Escatol/química , Escatol/metabolismo , TermodinâmicaRESUMO
BACKGROUND: Structure-based methods for P450 substrates are commonly used during drug development to identify sites of metabolism. However, docking studies using available X-ray structures for the major drug-metabolizing P450, CYP3A4, do not always identify binding modes supportive of the production of high-energy toxic metabolites. Minor pathways such as P450-catalyzed dehydrogenation have been experimentally shown to produce reactive products capable of forming biomolecular adducts which can lead to increased risk toxicities. 4-Hydroxy-tamoxifen (4OHT) is metabolized by CYP3A4 via competing hydroxylation and dehydrogenation reactions. METHODS: Ab initio gas-phase electronic structural characterization of 4OHT was used to develop a docking scoring scheme. Conformational sampling of CYP3A4 with molecular dynamics simulations along multiple trajectories were used to generate representative structures for docking studies using recently published heme parameters. A key predicted binding mode was tested experimentally using site-directed mutagenesis of CYP3A4 and liquid chromatography-mass spectroscopy analysis. RESULTS: Docking with MD-refined CYP3A4 structures incorporating hexa-coordinate heme parameters identifies a unique binding mode involving ARG212 and channel 4, unobserved in the starting PDB ID: 1TQN X-ray structure. The models supporting dehydrogenation are consistent with results from in vitro incubations. GENERAL SIGNIFICANCE: Our models indicate that coupled structural contributions of the ingress, egress and solvent channels to the CYP3A4 active site geometries play key roles in the observed 4OHT binding modes. Thus adequate sampling of the conformational space of these drug-metabolizing promiscuous enzymes is important for substrates that may bind in malleable regions of the enzyme active-site.
Assuntos
Arginina/fisiologia , Citocromo P-450 CYP3A/química , Citocromo P-450 CYP3A/metabolismo , Domínios e Motivos de Interação entre Proteínas/fisiologia , Tamoxifeno/análogos & derivados , Arginina/genética , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/fisiologia , Humanos , Hidrogenação , Ativação do Canal Iônico/genética , Ativação do Canal Iônico/fisiologia , Modelos Biológicos , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Solventes/metabolismo , Tamoxifeno/química , Tamoxifeno/metabolismo , Tamoxifeno/farmacocinéticaRESUMO
The aim of this study was to determine whether mouse CYP2A5 and CYP2F2 play critical roles in the bioactivation of 3-methylindole (3MI), a tissue-selective toxicant, in the target tissues, the nasal olfactory mucosa (OM) and lung. Five metabolites of 3MI were identified in NADPH- and GSH-fortified microsomal reactions, including 3-glutathionyl-S-methylindole (GS-A1), 3-methyl-2-glutathionyl-S-indole (GS-A2), 3-hydroxy-3-methyleneindolenine (HMI), indole-3-carbinol (I-3-C), and 3-methyloxindole (MOI). The metabolite profiles and enzyme kinetics of the reactions were compared between OM and lung, and among wild-type, Cyp2a5-null, and Cyp2f2-null mice. In lung reactions, GS-A1, GS-A2, and HMI were detected as major products, and I-3-C and MOI, as minor metabolites. In OM reactions, all five metabolites were detected in ample amounts. The loss of CYP2F2 affected formation of all 3MI metabolites in the lung and formation of HMI, GS-A1, and GS-A2 in the OM. In contrast, loss of CYP2A5 did not affect formation of 3MI metabolites in the lung but caused substantial decreases in I-3-C and MOI formation in the OM. Thus, whereas CYP2F2 plays a critical role in the 3MI metabolism in the lung, both CYP2A5 and CYP2F2 play important roles in 3MI metabolism in the OM. Furthermore, the fate of the reactive metabolites produced by the two enzymes through common dehydrogenation and epoxidation pathways seemed to differ with CYP2A5 supporting direct conversion to stable metabolites and CYP2F2 supporting further formation of reactive iminium ions. These results provide the basis for understanding the respective roles of CYP2A5 and CYP2F2 in 3MI's toxicity in the respiratory tract.
Assuntos
Hidrocarboneto de Aril Hidroxilases/fisiologia , Sistema Enzimático do Citocromo P-450/fisiologia , Pulmão/metabolismo , Mucosa Olfatória/metabolismo , Escatol/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/genética , Biotransformação , Cromatografia Líquida de Alta Pressão , Citocromo P-450 CYP2A6 , Sistema Enzimático do Citocromo P-450/genética , Família 2 do Citocromo P450 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microssomos/metabolismo , Escatol/farmacocinética , Escatol/toxicidade , Espectrometria de Massas em TandemRESUMO
Endogenous agonists of transient receptor potential vanilloid-1 (TRPV1) (endovanilloids) are implicated as mediators of lung injury during inflammation. This study tested the hypothesis that endovanilloids produced following lipopolysaccharide (LPS) treatment activate TRPV1 and cause endoplasmic reticulum stress/GADD153 expression in lung cells, representing a mechanistic component of lung injury. The TRPV1 agonist nonivamide induced GADD153 expression and caused cytotoxicity in immortalized and primary human bronchial, bronchiolar/alveolar, and microvascular endothelial cells, proportional to TRPV1 mRNA expression. In CF-1 mice, Trpv1 mRNA was most abundant in the alveoli, and intratracheal nonivamide treatment promoted Gadd153 expression in the alveolar region. Treatment of CF-1 mice with LPS increased Gadd153 in the lung, lactate dehydrogenase (LDH) in bronchoalveolar lavage (BAL) fluid, and lung wet-to-dry weight ratio. Cotreating mice with LPS and the TRPV1 antagonist LJO-328 reduced Gadd153 induction and LDH in BAL but did not inhibit increases in lung wet-to-dry ratio. In Trpv1(-/-) mice treated with LPS, Gadd153 induction and LDH in BAL were reduced relative to wild-type mice, and the wet-to-dry weight ratios of lungs from both wild-type and Trpv1(-/-) mice decreased. Organic extracts of blood collected from LPS-treated mice were more cytotoxic to TRPV1-overexpressing cells compared with BEAS-2B cells and extracts from control mice, however, most pure endovanilloids did not produce cytotoxicity in a characteristic TRPV1-dependent manner. Collectively, these data indicate a role for TRPV1, and endogenous TRPV1 agonists, in ER stress and cytotoxicity in lung cells but demonstrate that ER stress and cytotoxicity are not essential for pulmonary edema.
Assuntos
Estresse do Retículo Endoplasmático/genética , Lesão Pulmonar/fisiopatologia , Pulmão , Alvéolos Pulmonares/metabolismo , Canais de Cátion TRPV/agonistas , Animais , Brônquios/metabolismo , Líquido da Lavagem Broncoalveolar/química , Capsaicina/análogos & derivados , Capsaicina/farmacologia , Morte Celular , Linhagem Celular , Linhagem Celular Transformada , Células Cultivadas , Humanos , Inflamação/metabolismo , L-Lactato Desidrogenase/metabolismo , Lipopolissacarídeos/farmacologia , Pulmão/citologia , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Edema Pulmonar/metabolismo , Canais de Cátion TRPV/genética , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismoRESUMO
Molecular mechanics (MM) methods are computationally affordable tools for screening chemical libraries of novel compounds for sites of P450 metabolism. One challenge for MM methods has been the absence of a consistent and transferable set of parameters for the heme within the P450 active site. Experimental data indicate that mammalian P450 enzymes vary greatly in the size, architecture, and plasticity of their active sites. Thus, obtaining X-ray-based geometries for the development of accurate MM parameters for the major classes of hepatic P450 remains a daunting task. Our previous work with preliminary gas-phase quantum mechanics (QM)-derived atomic partial charges greatly improved the accuracy of docking studies of raloxifene to CYP3A4. We have therefore developed and tested a consistent set of transferable MM parameters based on gas-phase QM calculations of two model systems of the heme-a truncated (T-HM) and a full (F-HM) for four states of the P450 catalytic cycle. Our results indicate that the use of the atomic partial charges from the F-HM further improves the accuracy of docked predictions for raloxifene to CYP3A4. Different patterns for substrate docking are also observed depending on the choice of heme model and state. Newly parameterized heme models are tested in implicit and explicitly solvated MD simulations in the absence and presence of enzyme structures, for CYP3A4, and appear to be stable on the nanosecond simulation timescale. The new force field for the various heme states may aid the community for simulations of P450 enzymes and other heme-containing enzymes.
Assuntos
Biocatálise , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Heme/química , Teoria Quântica , Heme/metabolismo , Modelos Moleculares , Simulação de Dinâmica MolecularRESUMO
Environmental particulate matter (PM) pollutants adversely affect human health, but the molecular basis is poorly understood. The ion channel transient receptor potential vanilloid-1 (TRPV1) has been implicated as a sensor for environmental PM and a mediator of adverse events in the respiratory tract. The objectives of this study were to determine whether TRPV1 can distinguish chemically and physically unique PM that represents important sources of air pollution; to elucidate the molecular basis of TRPV1 activation by PM; and to ascertain the contributions of TRPV1 to human lung cell and mouse lung tissue responses exposed to an insoluble PM agonist, coal fly ash (CFA1). The major findings of this study are that TRPV1 is activated by some, but not all of the prototype PM materials evaluated, with rank-ordered responses of CFA1 > diesel exhaust PM > crystalline silica; TRP melastatin-8 is also robustly activated by CFA1, whereas other TRP channels expressed by airway sensory neurons and lung epithelial cells that may also be activated by CFA1, including TRPs ankyrin 1 (A1), canonical 4α (C4α), M2, V2, V3, and V4, were either slightly (TRPA1) or not activated by CFA1; activation of TRPV1 by CFA1 occurs via cell surface interactions between the solid components of CFA1 and specific amino acid residues of TRPV1 that are localized in the putative pore-loop region; and activation of TRPV1 by CFA1 is not exclusive in mouse lungs but represents a pathway by which CFA1 affects the expression of selected genes in lung epithelial cells and airway tissue.
Assuntos
Cinza de Carvão/toxicidade , Pulmão/efeitos dos fármacos , Canais de Cátion TRPV/fisiologia , Sequência de Bases , Linhagem Celular Transformada , Primers do DNA , Perfilação da Expressão Gênica , Humanos , Pulmão/metabolismo , Reação em Cadeia da PolimeraseRESUMO
Inhalation of environmental particulate matter (PM) is correlated with adverse health effects in humans, but gene products that couple detection with cellular responses, and the specific properties of PM that target different pathways, have not been fully elucidated. TRPA1 and V1 are two cation channels expressed by sensory neurons and non-neuronal cells of the respiratory tract that have been implicated as possible mediators of PM toxicity. The goals of this research were to determine if environmental PM preferentially activated TRPA1 and to elucidate the criteria responsible for selectivity. Quantification of TRPA1 activation by 4 model PM revealed that diesel exhaust PM (DEP) and coal fly ash PM (CFA1) were TRPA1 agonists at concentrations >0.077 mg/mL. DEP was more potent, and approximately 97% of the activity of DEP was recovered by serial extraction of the solid DEP with ethanol and hexane/n-butyl chloride. Modification of the electrophile/agonist binding sites on TRPA1 (C621, C641, C665, and K710) to non-nucleophilic residues reduced TRPA1 activation by DEP and abolished activation by DEP extracts as well as multiple individual electrophilic chemical components of DEP. However, responses to CFA1 and DEP solids were not affected by these mutations. Activity-guided fractionation of DEP and high resolution mass spectroscopy identified several new DEP-derived TRPA1 agonists, and activation of mouse dorsal root ganglion neurons demonstrated that TRPA1 is a primary target for DEP in a heterogeneous population of primary sensory nerves. It is concluded that TRPA1 is a specific target for electrophilic chemical components of DEP and proposed that activation of TRPA1 in the respiratory tract is likely to be an important mechanism for DEP pneumotoxicity.
Assuntos
Canais de Cálcio/metabolismo , Carbono/toxicidade , Gânglios Espinais/citologia , Pneumopatias/induzido quimicamente , Proteínas do Tecido Nervoso/metabolismo , Material Particulado/toxicidade , Canais de Potencial de Receptor Transitório/metabolismo , Emissões de Veículos/toxicidade , Animais , Canais de Cálcio/genética , Linhagem Celular , Células Cultivadas , Cinza de Carvão , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Canal de Cátion TRPA1 , Canais de Potencial de Receptor Transitório/agonistas , Canais de Potencial de Receptor Transitório/genéticaRESUMO
Activation of intracellular transient receptor potential vanilloid-1 (TRPV1) in human lung cells causes endoplasmic reticulum (ER) stress, increased expression of proapoptotic GADD153 (growth arrest- and DNA damage-inducible transcript 3), and cytotoxicity. However, in cells with low TRPV1 expression, cell death is not inhibited by TRPV1 antagonists, despite preventing GADD153 induction. In this study, chemical variants of the capsaicin analog nonivamide were synthesized and used to probe the relationship between TRPV1 receptor binding, ER calcium release, GADD153 expression, and cell death in TRPV1-overexpressing BEAS-2B, normal BEAS-2B, and primary normal human bronchial epithelial lung cells. Modification of the 3-methoxy-4-hydroxybenzylamide vanilloid ring pharmacophore of nonivamide reduced the potency of the analogs and rendered several analogs mildly inhibitory. Correlation analysis of analog-induced calcium flux, GADD153 induction, and cytotoxicity revealed a direct relationship for all three endpoints in all three lung cell types for nonivamide and N-(3,4-dihydroxybenzyl)nonanamide. However, the N-(3,4-dihydroxybenzyl)nonanamide analog also produced cytotoxicity through redox cycling/reactive oxygen species formation, shown by inhibition of cell death by N-acetylcysteine. Molecular modeling of binding interactions between the analogs and TRPV1 agreed with data for reduced potency of the analogs, and only nonivamide was predicted to form a "productive" ligand-receptor complex. This study provides vital information on the molecular interactions of capsaicinoids with TRPV1 and substantiates TRPV1-mediated ER stress as a conserved mechanism of lung cell death by prototypical TRPV1 agonists.
Assuntos
Capsaicina/análogos & derivados , Capsaicina/farmacologia , Células Epiteliais/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Canais de Cátion TRPV/efeitos dos fármacos , Cálcio/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fluorometria , Humanos , Pulmão/citologia , Modelos Moleculares , RNA/biossíntese , RNA/genética , Mucosa Respiratória/citologia , Mucosa Respiratória/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Relação Estrutura-Atividade , Fator de Transcrição CHOP/biossíntese , Fator de Transcrição CHOP/genéticaRESUMO
The use of molecular modeling in conjunction with site-directed mutagenesis has been extensively used to study substrate orientation within cytochrome P450 active sites and to identify potential residues involved in the positioning and catalytic mechanisms of these substrates. However, because docking studies utilize static models to simulate dynamic P450 enzymes, the effectiveness of these studies is strongly dependent on accurate enzyme models. This study employed a cytochrome P450 3A4 (CYP3A4) crystal structure (Protein Data Bank entry 1W0E) to predict the sites of metabolism of the known CYP3A4 substrate raloxifene. In addition, partial charges were incorporated into the P450 heme moiety to investigate the effect of the modified CYP3A4 model on metabolite prediction with the ligand docking program Autodock. Dehydrogenation of raloxifene to an electrophilic diquinone methide intermediate has been linked to the potent inactivation of CYP3A4. Active site residues involved in the positioning and/or catalysis of raloxifene supporting dehydrogenation were identified with the two models, and site-directed mutagenesis studies were conducted to validate the models. The addition of partial charges to the heme moiety improved the accuracy of the docking studies, increasing the number of conformations predicting dehydrogenation and facilitating the identification of substrate-active site residue interactions. On the basis of the improved model, the Phe215 residue was hypothesized to play an important role in orienting raloxifene for dehydrogenation through a combination of electrostatic and steric interactions. Substitution of this residue with glycine or glutamine significantly decreased dehydrogenation rates without concurrent changes in the rates of raloxifene oxygenation. Thus, the improved structural model predicted novel enzyme-substrate interactions that control the selective dehydrogenation of raloxifene to its protein-binding intermediate.
Assuntos
Citocromo P-450 CYP3A/química , Heme/química , Modelos Moleculares , Fenilalanina/química , Cloridrato de Raloxifeno/química , Substituição de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Ativação Enzimática/genética , Heme/genética , Heme/metabolismo , Humanos , Fenilalanina/genética , Fenilalanina/metabolismo , Ligação Proteica , Cloridrato de Raloxifeno/metabolismoRESUMO
Inhaled glucocorticoid (GC) therapy is a vital part of the management of chronic asthma. GCs are metabolized by members of the cytochrome P450 3A family in both liver and lung, but the enzymes are differentially expressed. Selective inhibition of one or more P450 3A enzymes could substantially modify target and systemic concentrations of GCs. In this study, we have evaluated the mechanism-based inactivation of P450 3A4, 3A5, and 3A7 enzymes by GCs. Among the five major inhaled GCs approved for clinical use in the United States, fluticasone propionate (FLT) was the most potent mechanism-based inactivator of P450 3A5, the predominant P450 enzyme in the lung. FLT inactivated P450 3A5 in a time- and concentration-dependent manner with K(I), k(inact), and partition ratio of 16 muM, 0.027 min(-1), and 3, respectively. In contrast, FLT minimally inactivated P450 3A4 and did not inactivate 3A7, even with a concentration of 100 muM. The inactivation of P450 3A5 by FLT was irreversible because dialysis did not restore enzyme activity. In addition, the exogenous nucleophilic scavenger GSH did not attenuate inactivation. The prosthetic heme of P450 3A5 was not modified by FLT. The loss of P450 3A5 activity in lung cells could substantially decrease the metabolism of FLT, which would increase the effective FLT concentration at its target site, the respiratory epithelium. Also, inactivation of lung P450 3A5 could increase the absorption of inhaled FLT, which could lead to high systemic concentrations and adverse effects, such as life-threatening adrenal crises or cataracts that have been documented in children receiving high doses of inhaled GCs.
Assuntos
Androstadienos/administração & dosagem , Androstadienos/farmacologia , Inibidores do Citocromo P-450 CYP3A , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacologia , Glucocorticoides/farmacologia , Pulmão/enzimologia , Administração por Inalação , Androstadienos/toxicidade , Antialérgicos/administração & dosagem , Antialérgicos/farmacologia , Antialérgicos/toxicidade , Citocromo P-450 CYP3A , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/toxicidade , Fluticasona , Glucocorticoides/administração & dosagem , Glucocorticoides/toxicidade , Humanos , Pulmão/citologia , Proteínas Recombinantes/antagonistas & inibidores , Relação Estrutura-Atividade , Fatores de TempoRESUMO
3-Methylindole (3MI) is a preferential pneumotoxicant found in cigarette smoke. A number of lung-expressed human cytochrome P450 enzymes, including 1A1, 2F1, and 2A13, catalyze the metabolism of 3MI to reactive intermediates that fragment DNA, measured with the Comet assay to assess DNA damage, in a cytochrome P450-dependent manner in primary normal human lung cells in culture, but the mutagenesis of 3MI has been controversial. In the present study, the mutagenic potential of 3MI was compared to the prototypical cigarette smoke carcinogens benzo(a)pyrene (B(a)P) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). 3MI, B(a)P, and NNK were incubated with the Salmonella typhimurium strain TA98, which is known to detect the most common subtype of cigarette smoke-induced mutagenicity, frameshift mutations in DNA, and with Salmonella typhimurium strain TA100, which detects base pair substitution mutants, with five sources of P450-mediated bioactivation: rat liver S9, human lung microsomes, recombinant CYP2A13, purified CYP2F3, and recombinant CYP1A1. Only B(a)P was mutagenic in TA100, and it was bioactivated by human lung microsomes and rat liver S9 sources of P450s. However, with the TA98 strain, CYP1A1, CYP2A13, CYP2F3, and human lung microsomes bioactivated 3MI to highly mutagenic intermediates, whereas neither human nor rat liver S9 subcellular fractions formed mutagenic intermediates from 3MI. Quantitative Western blot analysis verified that all three respiratory enzymes were present in human lung microsomes in widely varying amounts. These results indicate that metabolism of 3MI by human lung-expressed cytochrome P450 enzymes but not hepatic P450s elicits equivalent or higher mutagenicity than the prototype cigarette smoke mutagens B(a)P and NNK and indicates that 3MI is a likely human pulmonary carcinogen.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Pulmão/enzimologia , Mutagênicos/toxicidade , Escatol/toxicidade , Animais , Hidrocarboneto de Aril Hidroxilases/metabolismo , Benzo(a)pireno/química , Benzo(a)pireno/toxicidade , Células Cultivadas , Sistema Enzimático do Citocromo P-450/genética , Dano ao DNA , Humanos , Fígado/metabolismo , Microssomos/enzimologia , Microssomos/metabolismo , Testes de Mutagenicidade , Mutagênicos/química , Nitrosaminas/química , Nitrosaminas/toxicidade , Ratos , Proteína S9 Ribossômica , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Salmonella typhimurium/efeitos dos fármacos , Escatol/química , FumarRESUMO
Raloxifene was approved in 2007 by the FDA for the chemoprevention of breast cancer in postmenopausal women at high risk for invasive breast cancer. Approval was based in part on the improved safety profile for raloxifene relative to the standard treatment of tamoxifen. However, recent studies have demonstrated the ability of raloxifene to form reactive intermediates and act as a mechanism-based inhibitor of cytochrome P450 3A4 (CYP3A4) by forming adducts with the apoprotein. However, previous studies could not differentiate between dehydrogenation to a diquinone methide and the more common oxygenation pathway to an arene oxide as the most likely intermediate to inactivate CYP3A4. In the current work, (18)O-incorporation studies were utilized to carefully elucidate CYP3A4-mediated oxygenation versus dehydrogenation of raloxifene. These studies established that 3'-hydroxyraloxifene is produced exclusively via CYP3A4-mediated oxygenation and provide convincing evidence for the mechanism of CYP3A4-mediated dehydrogenation of raloxifene to a reactive diquinone methide, while excluding the alternative arene oxide pathway. Furthermore, it was demonstrated that 7-hydroxyraloxifene, which was previously believed to be a typical O(2)-derived metabolite of CYP3A4, is in fact produced by a highly unusual hydrolysis pathway from a putative ester, formed by the conjugation of raloxifene diquinone methide with a carboxylic acid moiety of CYP3A4, or other proteins in the reconstituted system. These findings not only confirm CYP3A4-mediated dehydrogenation of raloxifene to a reactive diquinone methide but also suggest a novel route of raloxifene toxicity.
Assuntos
Apoproteínas/metabolismo , Quimioprevenção , Citocromo P-450 CYP3A/metabolismo , Cloridrato de Raloxifeno/uso terapêutico , Tamoxifeno/uso terapêutico , Apoproteínas/farmacologia , Apoproteínas/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/prevenção & controle , Dano ao DNA , Feminino , Humanos , Cloridrato de Raloxifeno/metabolismo , Cloridrato de Raloxifeno/farmacologia , Tamoxifeno/metabolismo , Tamoxifeno/farmacologiaRESUMO
3-Methylindole (3MI) is a highly selective pneumotoxicant that is present in abundant amounts (as high as 1.4 mug/cigarette) in cigarette smoke. Several human cytochrome P450 enzymes that are expressed in lung, such as CYP1A1, CYP2F1, CYP2A13, and CYP4B1, catalyze the dehydrogenation of 3MI to the reactive intermediate 3-methyleneindolenine, which alkylates DNA and induces cell death through apoptosis. In addition, 3MI potently damages DNA at low concentrations (observable at 0.1 muM). However, it seemed possible that 3MI could induce the levels of P450 enzymes, so transcription and translation of 1A1 and 2F1 genes were measured in primary normal human bronchial epithelial cells. In this study, 3MI-induced DNA damage at the 10 muM concentration was ameliorated when P450 turnover was inactivated with the cytochrome P450 suicide substrate inhibitor 1-aminobenzotriazole. Thus, the observed DNA damage was cytochrome P450-dependent. Quantitative real-time polymerase chain reaction analysis revealed both concentration- and time-dependent increases in CYP1A1 and CYP2F1 transcription by the same 3MI concentrations that damaged DNA. Aryl hydrocarbon receptor (AhR) activation lead to CYP1A1 induction. Treatment with 3MI in combination with the AhR antagonist alpha-naphthoflavone prevented 3MI-mediated CYP1A1 induction, indicating that the induction was AhR-dependent. Conversely, CYP2F1 induction did not appear to require activation of AhR. These intriguing findings show that not only is induction of 1A1 and 2F1 caused by 3MI metabolites, rather than 3MI itself, but transcriptional activation of these pulmonary genes occurs through disparate mechanisms. Thus, the induction process, and subsequent increased bioactivation of 3MI to toxic intermediates, is a facile process that might enhance the acute toxicity and/or mutagenicity of this chemical.