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INTRODUCTION: The Population Assessment of Tobacco and Health (PATH) Study is a longitudinal cohort study on tobacco use behavior, attitudes and beliefs, and tobacco-related health outcomes, including biomarkers of tobacco exposure in the U.S. population. In this report we provide a summary of urinary nicotine metabolite measurements among adult users and non-users of tobacco from Wave 1 (2013-2014) of the PATH Study. METHODS: Total nicotine and its metabolites including cotinine, trans-3'-hydroxycotinine (HCTT), and other minor metabolites were measured in more than 11 500 adult participants by liquid chromatography tandem mass spectrometry methods. Weighted geometric means (GM) and least square means from statistical modeling were calculated for non-users and users of various tobacco products. RESULTS: Among daily users, the highest GM concentrations of nicotine, cotinine and HCTT were found in exclusive smokeless tobacco users, and the lowest in exclusive e-cigarette users. Exclusive combustible product users had intermediate concentrations, similar to those found in users of multiple products (polyusers). Concentrations increased with age within the categories of tobacco users, and differences associated with gender, race/ethnicity and educational attainment were also noted among user categories. Recent (past 12 months) former users had GM cotinine concentrations that were more than threefold greater than never users. CONCLUSIONS: These urinary nicotine metabolite data provide quantification of nicotine exposure representative of the entire US adult population during 2013-2014 and may serve as a reference for similar analyses in future measurements within this study. IMPLICATIONS: Nicotine and its metabolites in urine provide perhaps the most fundamental biomarkers of recent nicotine exposure. This report, based on Wave 1 of the Population Assessment of Tobacco and Health (PATH) Study, provides the first nationally representative data describing urinary nicotine biomarker concentrations in both non-users, and users of a variety of tobacco products including combustible, e-cigarette and smokeless products. These data provide a urinary biomarker concentration snapshot in time for the entire US population during 2013-2014, and will provide a basis for comparison with future results from continuing, periodic evaluations in the PATH Study.
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Sistemas Electrónicos de Liberación de Nicotina , Nicotina , Adulto , Biomarcadores/orina , Cotinina , Humanos , Estudios Longitudinales , Nicotina/orina , Autoinforme , Nicotiana , Uso de Tabaco/epidemiología , Uso de Tabaco/orinaRESUMEN
Electrophilic reactive intermediates resulting from drug metabolism have been associated with toxicity and off-target effects and in some drug discovery programs trigger NO-GO decisions. Many botanicals and dietary supplements are replete with such reactive electrophiles, notably Michael acceptors, which have been demonstrated to elicit chemopreventive mechanisms; and Michael acceptors are gaining regulatory approval as contemporary cancer therapeutics. Identifying protein targets of these electrophiles is central to understanding potential therapeutic benefit and toxicity risk. NO-donating NSAID prodrugs (NO-NSAIDs) have been the focus of extensive clinical and preclinical studies in inflammation and cancer chemoprevention and therapy: a subset exemplified by pNO-ASA, induces chemopreventive mechanisms following bioactivation to an electrophilic quinone methide (QM) Michael acceptor. Having previously shown that these NO-independent, QM-donors activated Nrf2 via covalent modification of Keap-1, we demonstrate that components of canonical NF-κB signaling are also targets, leading to the inhibition of NF-κB signaling. Combining bio-orthogonal probes of QM-donor ASA prodrugs with mass spectrometric proteomics and pathway analysis, we proceeded to characterize the quinonome: the protein cellular targets of QM-modification by pNO-ASA and its ASA pro-drug congeners. Further comparison was made using a biorthogonal probe of the "bare-bones", Michael acceptor, and clinical anti-inflammatory agent, dimethyl fumarate, which we have shown to inhibit NF-κB signaling. Identified quinonome pathways include post-translational protein folding, cell-death regulation, protein transport, and glycolysis; and identified proteins included multiple heat shock elements, the latter functionally confirmed by demonstrating activation of heat shock response.
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FN-kappa B/metabolismo , Profármacos/farmacocinética , Quinonas/farmacocinética , Activación Metabólica , Células HT29 , Humanos , Espectrometría de Masas , Factor 2 Relacionado con NF-E2/metabolismo , Proteómica , Teoría CuánticaRESUMEN
BACKGROUND: Synaptic dysfunction is a key event in pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD) where synapse loss pathologically correlates with cognitive decline and dementia. Although evidence suggests that aberrant protein production and aggregation are the causative factors in familial subsets of such diseases, drugs singularly targeting these hallmark proteins, such as amyloid-ß, have failed in late stage clinical trials. Therefore, to provide a successful disease-modifying compound and address synaptic dysfunction and memory loss in AD and mixed pathology dementia, we repurposed a clinically proven drug, CMZ, with neuroprotective and anti-inflammatory properties via addition of nitric oxide (NO) and cGMP signaling property. RESULTS: The novel compound, NMZ, was shown to retain the GABAA potentiating actions of CMZ in vitro and sedative activity in vivo. Importantly, NMZ restored LTP in hippocampal slices from AD transgenic mice, whereas CMZ was without effect. NMZ reversed amnestic blockade of acetylcholine receptors by scopolamine as well as NMDA receptor blockade by a benzodiazepine and a NO synthase inhibitor in the step-through passive avoidance (STPA) test of learning and working memory. A PK/PD relationship was developed based on STPA analysis coupled with pharmacokinetic measures of drug levels in the brain: at 1 nM concentration in brain and plasma, NMZ was able to restore memory consolidation in mice. CONCLUSION: Our findings show that NMZ embodies a promising pharmacological approach targeting synaptic dysfunction and opens new avenues for neuroprotective intervention strategies in mixed pathology AD, neurodegeneration, and dementia.
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Enfermedad de Alzheimer/tratamiento farmacológico , Clormetiazol/análogos & derivados , Reposicionamiento de Medicamentos/métodos , Agonistas de Receptores de GABA-A/farmacología , Hipocampo/efectos de los fármacos , Potenciación a Largo Plazo/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Nootrópicos/farmacología , Animales , Proteína de Unión a CREB/metabolismo , GMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Agonistas de Receptores de GABA-A/farmacocinética , Masculino , Ratones , Ratones Transgénicos , Fármacos Neuroprotectores/farmacocinética , Óxido Nítrico/metabolismo , Nootrópicos/farmacocinética , Transducción de Señal/efectos de los fármacos , Sinapsis/efectos de los fármacos , Sinapsis/patología , Xenopus laevisRESUMEN
The fruit fly (Drosophila melanogaster) is an extensively used and powerful, genetic model organism. However, chemical studies using individual flies have been limited by the animal's small size. Introduced here is a method to sample nanoliter hemolymph volumes from individual adult fruit-flies for chemical analysis. The technique results in an ability to distinguish hemolymph chemical variations with developmental stage, fly sex, and sampling conditions. Also presented is the means for two-point monitoring of hemolymph composition for individual flies.
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Electroforesis Capilar , Hemolinfa/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Aminoácidos/química , Animales , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Femenino , Larva/química , Masculino , Nanotecnología , Factores SexualesRESUMEN
The promising therapeutic potential of the NO-donating hybrid aspirin prodrugs (NO-ASA) includes induction of chemopreventive mechanisms and has been reported in almost 100 publications. One example, NCX-4040 (pNO-ASA), is bioactivated by esterase to a quinone methide (QM) electrophile. In cell cultures, pNO-ASA and QM-donating X-ASA prodrugs that cannot release NO rapidly depleted intracellular GSH and caused DNA damage; however, induction of Nrf2 signaling elicited cellular defense mechanisms including upregulation of NAD(P)H:quinone oxidoreductase-1 (NQO1) and glutamate-cysteine ligase (GCL). In HepG2 cells, the "NO-specific" 4,5-diaminofluorescein reporter, DAF-DA, responded to NO-ASA and X-ASA, with QM-induced oxidative stress masquerading as NO. LC-MS/MS analysis demonstrated efficient alkylation of Cys residues of proteins including glutathione-S-transferase-P1 (GST-P1) and Kelch-like ECH-associated protein 1 (Keap1). Evidence was obtained for alkylation of Keap1 Cys residues associated with Nrf2 translocation to the nucleus, nuclear translocation of Nrf2, activation of antioxidant response element (ARE), and upregulation of cytoprotective target genes. At least in cell culture, pNO-ASA acts as a QM donor, bioactivated by cellular esterase activity to release salicylates, NO(3)(-), and an electrophilic QM. Finally, two novel aspirin prodrugs were synthesized, both potent activators of ARE, designed to release only the QM and salicylates on bioactivation. Current interest in electrophilic drugs acting via Nrf2 signaling suggests that QM-donating hybrid drugs can be designed as informative chemical probes in drug discovery.
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Aspirina/análogos & derivados , Aspirina/farmacología , Indolquinonas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Profármacos/farmacología , Animales , Línea Celular Tumoral , Daño del ADN , Glutatión/metabolismo , Gutatión-S-Transferasa pi/metabolismo , Humanos , Proteína 1 Asociada A ECH Tipo Kelch , Ratones , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Óxido Nítrico/metabolismoRESUMEN
Tobacco use is the leading preventable cause of premature disease and death in the United States. Approximately, 34 million U.S. adults currently smoke cigarettes. We developed a method for automated sample preparation and liquid chromatography-tandem mass spectrometry quantitation of 14 tobacco-related analytes: nicotine (NICF), cotinine (COTF), trans-3'-hydroxycotinine (HCTF), menthol glucuronide (MEG), anabasine (ANBF), anatabine (ANTF), isonicoteine (ISNT), myosmine (MYOS), beta-nicotyrine (BNTR), bupropion (BUPR), cytisine (CYTI), varenicline (VARE), arecaidine (ARD), and arecoline (ARL). The method includes automated solid-phase extraction using customized positive-pressure functions. The preparation scheme has the capacity to process a batch of 96 samples within 4 h with greater than 88% recovery for all analytes. The 14 analytes, separated within 4.15 min using reversed-phase liquid chromatography, were determined using a triple-quadrupole mass spectrometer with atmospheric-pressure chemical ionization and multiple reaction monitoring in negative and positive ionization modes. Wide quantitation ranges, within 1.2-72,000 ng/mL, were established especially for COTF, HCTF, MEG, and NICF to quantify the broad range of biomarker concentrations found in the U.S. population. The method accuracy is above 90% while the overall imprecision is below 7%. Finally, we tested urine samples from 90 smokers and observed detection rates of over 98% for six analytes with urinary HCTF and MEG concentrations ranging from 200-14,100 and 60-57,100 ng/mL, respectively. This high throughput analytical process can prepare and analyze a sample in 9 min and along with the 14-compound analyte panel can be useful for tobacco-exposure studies, in smoking-cessation programs, and for detecting changes in exposure related to tobacco products and their use.
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This study investigated the effect of different sampling environments on hemolymph amino acid content of individual Drosophila melanogaster larvae. Hemolymph was collected from individual third instar larvae under cold-anesthetized, awake, and stress conditions. Qualitative and quantitative hemolymph amino acid analyses were performed via capillary electrophoresis with laser-induced fluorescence detection. The hemolymph amino acid concentrations, particularly arginine, glutamate, and taurine, changed significantly depending on the prior-to-sample-collection environments. Hemolymph amino acid analyses of six different Drosophila genotypes including two control genotypes and four mutant alleles were also carried out. Two mutant genotypes with over and under expression of a putative cystine-glutamate exchanger subunit were significantly different from each other with respect to their hemolymph glutamate, glycine, lysine, and taurine levels. Hemolymph amino acid analyses of stressed larvae of two control and two mutant genotypes indicated that behavior-related hemolymph chemical changes are also genotype dependent.
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Aminoácidos/sangre , Conducta Animal/fisiología , Drosophila melanogaster/genética , Drosophila melanogaster/fisiología , Variación Genética , Hemolinfa/química , Estrés Fisiológico , Alelos , Sistemas de Transporte de Aminoácidos/genética , Aminoácidos/genética , Anestesia , Animales , Proteínas de Drosophila/genética , Electroforesis Capilar , Genotipo , Larva , Mutación , Estrés Fisiológico/genéticaRESUMEN
A low-volume automated injection system for the analysis of chemically complex, amino acid samples is presented. This system utilizes submicroliter sample volumes stored on a 75-mum inner diameter capillary. A pulse of positive pressure (82 kPa) is used to load nanoliter sample volumes into an in-house fabricated interface and onto a separation capillary. Residual sample solution in the interface is immediately washed away by a continuous transverse flow through the injection interface, yielding a sharp and reproducible sample plug. By performing multiple injections of a static sample, one may average the signals to yield a signal-to-noise ratio improvement of up to 4.07-fold for 20 injections compared with a theoretical maximum of a 4.47-fold improvement. Without interruption of the applied voltage, injections performed every 150 s were used to monitor the progress of the reaction of multiple amino acids with the fluorogenic dye 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde. Analysis of dialyzed clinical vitreous samples demonstrates the resolution and quantitation of arginine, lysine, leucine, glutamine, and glutamate. Observed levels are comparable with those of nonautomated injection methods and reports by others.
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Aminoácidos/análisis , Electroforesis Capilar/instrumentación , Electroforesis Capilar/métodos , Cuerpo Vítreo/química , Cuerpo Vítreo/metabolismo , Diálisis , HumanosRESUMEN
Reversible chemical modifications of protein cysteine residues by S-nitrosylation and S-oxidation are increasingly recognized as important regulatory mechanisms for many protein classes associated with cellular signaling and stress response. Both modifications may theoretically occur under cellular nitrosative or nitroxidative stress. Therefore, a proteomic isotope-coded approach to parallel, quantitative analysis of cysteome S-nitrosylation and S-oxidation was developed. Modifications of cysteine residues of (i) human glutathione-S-transferase P1-1 (GSTP1) and (ii) the schistosomiasis drug target thioredoxin glutathione reductase (TGR) were studied. Both S-nitrosylation (SNO) and S-oxidation to disulfide (SS) were observed for reactive cysteines, dependent on concentration of added S-nitrosocysteine (CysNO) and independent of oxygen. SNO and SS modifications of GSTP1 were quantified and compared for therapeutically relevant NO and HNO donors from different chemical classes, revealing oxidative modification for all donors. Observations on GSTP1 were extended to cell cultures, analyzed after lysis and in-gel digestion. Treatment of living neuronal cells with CysNO, to induce nitrosative stress, caused levels of S-nitrosylation and S-oxidation of GSTP1 comparable to those of cell-free studies. Cysteine modifications of PARK7/DJ-1, peroxiredoxin-2, and other proteins were identified, quantified, and compared to overall levels of protein S-nitrosylation. The new methodology has allowed identification and quantitation of specific cysteome modifications, demonstrating that nitroxidation to protein disulfides occurs concurrently with S-nitrosylation to protein-SNO in recombinant proteins and living cells under nitrosative stress.
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Cisteína/análogos & derivados , Disulfuros/metabolismo , Gutatión-S-Transferasa pi/metabolismo , Complejos Multienzimáticos/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Estrés Oxidativo/fisiología , Proteómica , S-Nitrosotioles/metabolismo , Línea Celular Tumoral , Cisteína/metabolismo , Cisteína/farmacología , Gutatión-S-Transferasa pi/genética , Humanos , Complejos Multienzimáticos/genética , NADH NADPH Oxidorreductasas/genética , Neuronas/efectos de los fármacos , Neuronas/enzimología , Neuronas/metabolismo , Óxido Nítrico/química , Donantes de Óxido Nítrico/química , Nitrosación , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Procesamiento Proteico-Postraduccional , S-Nitrosotioles/farmacologíaRESUMEN
The risk of developing hormone-dependent cancers with long-term exposure to estrogens is attributed both to proliferative, hormonal actions at the estrogen receptor (ER) and to chemical carcinogenesis elicited by genotoxic, oxidative estrogen metabolites. Nontumorigenic MCF-10A human breast epithelial cells are classified as ER(-) and undergo estrogen-induced malignant transformation. Selective estrogen receptor modulators (SERM), in use for breast cancer chemoprevention and for postmenopausal osteoporosis, were observed to inhibit malignant transformation, as measured by anchorage-independent colony growth. This chemopreventive activity was observed to correlate with reduced levels of oxidative estrogen metabolites, cellular reactive oxygen species (ROS), and DNA oxidation. The ability of raloxifene, desmethylarzoxifene (DMA), and bazedoxifene to inhibit this chemical carcinogenesis pathway was not shared by 4-hydroxytamoxifen. Regulation of phase II rather than phase I metabolic enzymes was implicated mechanistically: raloxifene and DMA were observed to upregulate sulfotransferase (SULT 1E1) and glucuronidase (UGT 1A1). The results support upregulation of phase II metabolism in detoxification of catechol estrogen metabolites leading to attenuated ROS formation as a mechanism for inhibition of malignant transformation by a subset of clinically important SERMs.
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Transformación Celular Neoplásica , Citoprotección/efectos de los fármacos , Estradiol/efectos adversos , Inactivación Metabólica/efectos de los fármacos , Glándulas Mamarias Humanas/efectos de los fármacos , Oxidantes/metabolismo , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Transformación Celular Neoplásica/inducido químicamente , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/metabolismo , Células Cultivadas , Humanos , Indoles/farmacología , Células MCF-7 , Glándulas Mamarias Humanas/metabolismo , Glándulas Mamarias Humanas/patología , Estrés Oxidativo/efectos de los fármacos , Piperidinas/farmacología , Clorhidrato de Raloxifeno/farmacología , Especies Reactivas de Oxígeno/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacología , Tiofenos/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Selective estrogen receptor modulators (SERMs) are effective therapeutics that preserve favorable actions of estrogens on bone and act as antiestrogens in breast tissue, decreasing the risk of vertebral fractures and breast cancer, but their potential in neuroprotective and procognitive therapy is limited by: 1) an increased lifetime risk of thrombotic events; and 2) an attenuated response to estrogens with age, sometimes linked to endothelial nitric oxide synthase (eNOS) dysfunction. Herein, three 3(rd) generation SERMs with similar high affinity for estrogen receptors (ERα, ERß) were studied: desmethylarzoxifene (DMA), FDMA, and a novel NO-donating SERM (NO-DMA). Neuroprotection was studied in primary rat neurons exposed to oxygen glucose deprivation; reversal of cholinergic cognitive deficit was studied in mice in a behavioral model of memory; long term potentiation (LTP), underlying cognition, was measured in hippocampal slices from older 3×Tg Alzheimer's transgenic mice; vasodilation was measured in rat aortic strips; and anticoagulant activity was compared. Pharmacologic blockade of GPR30 and NOS; denudation of endothelium; measurement of NO; and genetic knockout of eNOS were used to probe mechanism. Comparison of the three chemical probes indicates key roles for GPR30 and eNOS in mediating therapeutic activity. Procognitive, vasodilator and anticoagulant activities of DMA were found to be eNOS dependent, while neuroprotection and restoration of LTP were both shown to be dependent upon GPR30, a G-protein coupled receptor mediating estrogenic function. Finally, the observation that an NO-SERM shows enhanced vasodilation and anticoagulant activity, while retaining the positive attributes of SERMs even in the presence of NOS dysfunction, indicates a potential therapeutic approach without the increased risk of thrombotic events.
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Anticoagulantes/farmacología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Donantes de Óxido Nítrico/farmacología , Nootrópicos/farmacología , Piperidinas/farmacología , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Tiofenos/farmacología , Animales , Hipoxia de la Célula , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/metabolismo , Glucosa/deficiencia , Masculino , Memoria/efectos de los fármacos , Ratones , Neuronas/citología , Neuronas/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo III/metabolismo , Cultivo Primario de Células , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Receptores de Estrógenos , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/metabolismo , Vasodilatación/efectos de los fármacosRESUMEN
One of the most widely used transgenic animal models in biology is Drosophila melanogaster, the fruit fly. Chemical information from this exceedingly small organism is usually accomplished by studying populations to attain sample volumes suitable for standard analysis methods. This paper describes a direct sampling technique capable of obtaining 50-300 nL of hemolymph from individual Drosophila larvae. Hemolymph sampling performed under mineral oil and in air at 30 s intervals up to 120 s after piercing larvae revealed that the effect of evaporation on amino acid concentrations is insignificant when the sample was collected within 60 s. Qualitative and quantitative amino acid analyses of obtained hemolymph were carried out in two optimized buffer conditions by capillary electrophoresis with laser-induced fluorescence detection after derivatizing with fluorescamine. Thirteen amino acids were identified from individual hemolymph samples of both wild-type (WT) control and the genderblind (gb) mutant larvae. The levels of glutamine, glutamate, and taurine in the gb hemolymph were significantly lower at 35%, 38%, and 57% of WT levels, respectively. The developed technique that samples only the hemolymph fluid is efficient and enables accurate organism-level chemical information while minimizing errors associated with possible sample contaminations, estimations, and effects of evaporation compared to the traditional hemolymph-sampling techniques.