RESUMO
BACKGROUND: We previously demonstrated the in vitro killing of AML cells by the combination of the lipid-lowering agent bezafibrate (BEZ) and the contraceptive hormone medroxyprogesterone acetate (MPA). A phase II trial demonstrated in vivo safety and efficacy of BEZ and MPA (BaP) in elderly, relapsed/refractory AML and high-risk myelodysplastic syndrome (MDS) patients. However, we observed dose-limiting toxicities in a second trial that attempted to improve outcomes via escalation of BaP doses. Thus we sought to identify a third repurposed drug that potentiates activity of low dose BaP (BaP 0.1 mM). METHODS AND RESULTS: We demonstrate that addition of a commonly used anti-epileptic, valproic acid (VAL) to low dose BaP (BaP 0.1 mM)(VBaP) enhanced killing of AML cell lines/primary AML cells to levels similar to high dose BaP (BaP 0.5 mM). Similarly, addition of VAL to BaP 0.1 mM enhanced reactive oxygen species (ROS), lipid peroxidation and inhibition of de novo fatty acid synthesis. Overexpression of Nrf2 in K562 and KG1a completely inhibited ROS production and rescued cells from VAL/BaP 0.1 mM/VBaP killing. CONCLUSIONS: Given the good safety data of low-dose BaP in elderly/relapsed/refractory AML patients, and that VAL alone is well-tolerated, we propose VBaP as a novel therapeutic combination for AML.
Assuntos
Antioxidantes/metabolismo , Bezafibrato/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Acetato de Medroxiprogesterona/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ácido Valproico/farmacologia , Anticonvulsivantes/farmacologia , Linhagem Celular Tumoral , Contraceptivos Hormonais/farmacologia , Humanos , Hipolipemiantes/farmacologia , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Dose Máxima TolerávelRESUMO
Metabolic phenotyping of tissues uses metabolomics and lipidomics to measure the relative polar and nonpolar (lipid) metabolite levels in biological samples. This approach aims to understand disease biochemistry and identify biochemical markers of disease. Sample preparation methods must be reproducible, sensitive (high metabolite and lipid yield), and ideally rapid. We evaluated three biphasic methods for polar and nonpolar compound extraction (chloroform/methanol/water, dichloromethane/methanol/water, and methyl tert-butyl ether [MTBE]/methanol/water), a monophasic method for polar compound extraction (acetonitrile/methanol/water), and a monophasic method for nonpolar compound extraction (isopropanol/water). All methods were applied to mammalian heart, kidney, and liver tissues. Polar extracts were analyzed by hydrophilic interaction chromatography (HILIC) ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS) and nonpolar extracts by C18 reversed-phase UHPLC-MS. Method reproducibility and yield were assessed using multiple annotated endogenous compounds (putatively and MS/MS annotated). Monophasic methods had the highest yield and high reproducibility for both polar (positive ion: median relative standard deviation (RSD) < 18%; negative ion: median RSD < 28%) and nonpolar (positive and negative ion: median RSD < 15%) extractions for heart, kidneys, and liver. The polar monophasic method extracted higher levels of lipid than biphasic polar extractions, and these lipids caused minimal detection suppression for other compounds during HILIC UHPLC-MS. The nonpolar monophasic method had similar or greater detection responses of all detected lipid classes compared to biphasic methods (including increased phosphatidylinositol, phosphatidylserine, and cardiolipin responses). Monophasic methods are quicker and simpler than biphasic methods and are therefore most suited for future automation.
Assuntos
Lipídeos , Espectrometria de Massas em Tandem , Animais , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Reprodutibilidade dos Testes , SolventesRESUMO
INTRODUCTION: High plasma triacylglyceride levels are known to be associated with increased risk of atherosclerotic cardiovascular disease. Apolipoprotein C-III (apoC-III) is a key regulator of plasma triacylglyceride levels and is associated with hypertriglyceridemia via a number of pathways. There is consistent evidence for an association of cardiovascular events with blood apoC-III level, with support from human genetic studies of APOC3 variants. As such, apoC-III has been recognised as a potential therapeutic target for patients with severe hypertriglyceridaemia with one of the most promising apoC-III-targeting drugs, volanesorsen, having recently progressed through Phase III trials. OBJECTIVES: To exploit a rare loss of function variant in APOC3 (rs138326449) to characterise the potential long-term treatment effects of apoC-III targeting interventions on the metabolome. METHODS: In a recall-by-genotype study, 115 plasma samples were analysed by UHPLC-MS to acquire non-targeted metabolomics data. The study included samples from 57 adolescents and 33 adults. Overall, 12 985 metabolic features were tested for an association with APOC3 genotype. RESULTS: 161 uniquely annotated metabolites were found to be associated with rs138326449(APOC3). The highest proportion of associated metabolites belonged to the acyl-acyl glycerophospholipid and triacylglyceride metabolite classes. In addition to the anticipated (on-target) reduction of metabolites in the triacylglyceride and related classes, carriers of the rare variant exhibited previously unreported increases in levels of a number of metabolites from the acyl-alkyl glycerophospholipid class. CONCLUSION: Overall, our results suggest that therapies targeting apoC-III may potentially achieve a broad shift in lipid profile that favours better metabolic health.
Assuntos
Apolipoproteína C-III/genética , Apolipoproteína C-III/metabolismo , Adolescente , Adulto , Apolipoproteína C-III/sangue , Feminino , Genótipo , Humanos , Hipertrigliceridemia/metabolismo , Lipídeos/fisiologia , Lipoproteínas/metabolismo , Masculino , Metaboloma/fisiologia , Metabolômica , Triglicerídeos/sangue , Triglicerídeos/metabolismo , Triglicerídeos/uso terapêutico , Adulto JovemRESUMO
Clinical metabolic phenotyping employs metabolomics and lipidomics to detect and measure hundreds to thousands of metabolites and lipids within human samples. This approach aims to identify metabolite and lipid changes between phenotypes (e.g. disease status) that aid understanding of biochemical mechanisms driving the phenotype. Sample preparation is a critical step in clinical metabolic phenotyping: it must be reproducible and give a high extraction yield of metabolites and lipids, and in high-throughput studies it needs to be rapid. Here, we assessed the extraction of polar metabolites from human urine and polar metabolites and lipids from human plasma for analysis by ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) metabolomics and lipidomics. We evaluated several monophasic (urine and plasma) and biphasic (plasma) extractions, and we also tested alterations to (a) solvent-biofluid incubation time and temperature during monophasic extraction, and (b) phase partitioning time during biphasic extraction. Extracts were analysed by three UHPLC-MS assays: (i) hydrophilic interaction chromatography (HILIC) for urine and plasma, (ii) C18 aqueous reversed phase for urine, and (iii) C18 reversed phase for plasma lipids, and the yield and reproducibility of each method was assessed. We measured UHPLC-MS injection reproducibility as well as sample preparation reproducibility to assess sample solvent composition compatibility with UHPLC-MS and to pinpoint the origin of variance within the methods. For HILIC UHPLC-MS plasma and urine analysis, monophasic 50 : 50 methanol : acetonitrile had the most detected putatively-identified polar metabolites with high method reproducibility. This method had the highest lipid yield for plasma extracts analysed by the HILIC method. If lipid removal from the plasma polar HILIC extract is required, then the biphasic methanol/chloroform/water method is recommended. For C18 (aqueous) UHPLC-MS urine analysis, 50 : 50 methanol : water had high reproducibility and yield. For C18 UHPLC-MS plasma lipidomics, monophasic 100% isopropanol had the highest detection response of all annotated lipid classes with high reproducibility. Increasing monophasic incubation time and temperature had little benefit on metabolite and lipid yield and reproducibility for all methods.
Assuntos
Metabolômica , Cromatografia Líquida de Alta Pressão , Humanos , Espectrometria de Massas , Reprodutibilidade dos Testes , SolventesRESUMO
A challenge facing metabolomics in the analysis of large human cohorts is the cross-laboratory comparability of quantitative metabolomics measurements. In this study, 14 laboratories analyzed various blood specimens using a common experimental protocol provided with the Biocrates AbsoluteIDQ p400HR kit, to quantify up to 408 metabolites. The specimens included human plasma and serum from male and female donors, mouse and rat plasma, as well as NIST SRM 1950 reference plasma. The metabolite classes covered range from polar (e.g., amino acids and biogenic amines) to nonpolar (e.g., diacyl- and triacyl-glycerols), and they span 11 common metabolite classes. The manuscript describes a strict system suitability testing (SST) criteria used to evaluate each laboratory's readiness to perform the assay, and provides the SST Skyline documents for public dissemination. The study found approximately 250 metabolites were routinely quantified in the sample types tested, using Orbitrap instruments. Interlaboratory variance for the NIST SRM-1950 has a median of 10% for amino acids, 24% for biogenic amines, 38% for acylcarnitines, 25% for glycerolipids, 23% for glycerophospholipids, 16% for cholesteryl esters, 15% for sphingolipids, and 9% for hexoses. Comparing to consensus values for NIST SRM-1950, nearly 80% of comparable analytes demonstrated bias of <50% from the reference value. The findings of this study result in recommendations of best practices for system suitability, quality control, and calibration. We demonstrate that with appropriate controls, high-resolution metabolomics can provide accurate results with good precision across laboratories, and the p400HR therefore is a reliable approach for generating consistent and comparable metabolomics data.
Assuntos
Aminoácidos/sangue , Aminas Biogênicas/sangue , Análise Química do Sangue/estatística & dados numéricos , Lipidômica/estatística & dados numéricos , Lipídeos/sangue , Metabolômica/estatística & dados numéricos , Análise de Variância , Animais , Cromatografia Líquida de Alta Pressão/estatística & dados numéricos , Agregação de Dados , Feminino , Humanos , Limite de Detecção , Masculino , Espectrometria de Massas/estatística & dados numéricos , Metaboloma , Camundongos , Ratos , Reprodutibilidade dos TestesRESUMO
INTRODUCTION: Exposure to ricin can be lethal and treatments that are under development have short windows of opportunity for administration after exposure. It is therefore essential to achieve early detection of ricin exposure to provide the best prognosis for exposed individuals. Ricin toxin can be detected in clinical samples via several antibody-based techniques, but the efficacy of these can be limited due to the rapid processing and cellular uptake of toxin in the body and subsequent low blood ricin concentrations. Other diagnostic tools that perform, in an orthogonal manner, are therefore desirable. OBJECTIVES: To determine time-dependent metabolic changes in Sprague-Dawley rats following intravenous exposure to ricin. METHODS: Sprague-Dawley rats were intravenously exposed to ricin and multiple blood samples were collected from each animal for up to 48 h following exposure in two independent studies. Plasma samples were analysed applying HILIC and C18 reversed phase UHPLC-MS assays followed by univariate and multivariate analysis. RESULTS: In Sprague-Dawley rats we have demonstrated that metabolic changes measured in blood can distinguish between rats exposed intravenously to ricin and controls prior to the onset of behavioral signs of intoxication after 24 h. A total of 37 metabolites were significantly altered following exposure to ricin when compared to controls. The arginine/proline, bile acid and triacylglyceride metabolic pathways were highlighted as being important with two triacylglycerides at 8 h post exposure giving an AUROC score of 0.94. At 16 h and 24 h the AUROC score increased to 0.98 and 1.0 with the number of metabolites in the panel increasing to 5 and 7, respectively. CONCLUSIONS: These data demonstrate that metabolites may be a useful tool to diagnose and detect ricin exposure, thus increasing the effectiveness of supportive therapy and future ricin-specific medical treatments.
Assuntos
Substâncias para a Guerra Química/toxicidade , Metaboloma/efeitos dos fármacos , Metabolômica/métodos , Ricina/toxicidade , Animais , Área Sob a Curva , Arginina/metabolismo , Biomarcadores/sangue , Substâncias para a Guerra Química/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Meia-Vida , Masculino , Espectrometria de Massas , Redes e Vias Metabólicas , Modelos Animais , Curva ROC , Ratos , Ratos Sprague-Dawley , Ricina/metabolismo , Triglicerídeos/metabolismoRESUMO
Objective: The study objective was to determine whether adequately delivered bilateral remote ischemic preconditioning is cardioprotective in young children undergoing surgery for 2 common congenital heart defects with or without cyanosis. Methods: We performed a prospective, double-blind, randomized controlled trial at 2 centers in the United Kingdom. Children aged 3 to 36 months undergoing tetralogy of Fallot repair or ventricular septal defect closure were randomized 1:1 to receive bilateral preconditioning or sham intervention. Participants were followed up until hospital discharge or 30 days. The primary outcome was area under the curve for high-sensitivity troponin-T in the first 24 hours after surgery, analyzed by intention-to-treat. Right atrial biopsies were obtained in selected participants. Results: Between October 2016 and December 2020, 120 eligible children were randomized to receive bilateral preconditioning (n = 60) or sham intervention (n = 60). The primary outcome, area under the curve for high-sensitivity troponin-T, was higher in the preconditioning group (mean: 70.0 ± 50.9 µg/L/h, n = 56) than in controls (mean: 55.6 ± 30.1 µg/L/h, n = 58) (mean difference, 13.2 µg/L/h; 95% CI, 0.5-25.8; P = .04). Subgroup analyses did not show a differential treatment effect by oxygen saturations (pinteraction = .25), but there was evidence of a differential effect by underlying defect (pinteraction = .04). Secondary outcomes and myocardial metabolism, quantified in atrial biopsies, were not different between randomized groups. Conclusions: Bilateral remote ischemic preconditioning does not attenuate myocardial injury in children undergoing surgical repair for congenital heart defects, and there was evidence of potential harm in unstented tetralogy of Fallot. The routine use of remote ischemic preconditioning cannot be recommended for myocardial protection during pediatric cardiac surgery.
RESUMO
Interactions between epigenome and the environment in biology and in disease are of fundamental importance. The incidence of hepatocellular adenomas in flatfish exceeds 20% in some environments forming a unique opportunity to study environmental tumorigenesis of general relevance to cancer in humans. We report the novel finding of marked DNA methylation and metabolite concentration changes in histopathologically normal tissue distal to tumors in fish liver. A multi-"omics" discovery approach led to targeted and quantitative gene transcription analyses and metabolite analyses of hepatocellular adenomas and histologically normal liver tissue in the same fish. We discovered a remarkable and consistent global DNA hypomethylation, modification of DNA methylation and gene transcription, and disruption of one-carbon metabolism in distal tissue compared to livers of non-tumor-bearing fish. The mechanism of this disruption is linked not to depletion of S-adenosylmethionine, as is often a feature of mammalian tumors, but to a decrease in choline and elevated S-adenosylhomocysteine, a potent inhibitor of DNA methyltransferase. This novel feature of normal-appearing tissue of tumor-bearing fish helps to understand the unprecedentedly high incidence of tumors in fish sampled from the field and adds weight to the controversial epigenetic progenitor model of tumorigenesis. With further studies, the modifications may offer opportunities as biomarkers of exposure to environmental factors influencing disease.
Assuntos
Adenoma de Células Hepáticas/veterinária , Carcinogênese/genética , Metilação de DNA , Doenças dos Peixes/metabolismo , Neoplasias Hepáticas/veterinária , Fígado/metabolismo , S-Adenosil-Homocisteína/metabolismo , Adenoma de Células Hepáticas/genética , Adenoma de Células Hepáticas/metabolismo , Adenoma de Células Hepáticas/patologia , Animais , Carcinogênese/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Epigenômica , Doenças dos Peixes/genética , Doenças dos Peixes/patologia , Linguados , Regulação da Expressão Gênica , Interação Gene-Ambiente , Humanos , Fígado/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Microambiente TumoralRESUMO
Idiopathic intracranial hypertension, a disease classically occurring in women with obesity, is characterized by raised intracranial pressure. Weight loss leads to the reduction in intracranial pressure. Additionally, pharmacological glucagon-like peptide-1 agonism reduces cerebrospinal fluid secretion and intracranial pressure. The potential mechanisms by which weight loss reduces intracranial pressure are unknown and were the focus of this study. Meal stimulation tests (fasted plasma sample, then samples at 15, 30, 60, 90 and 120â min following a standardized meal) were conducted pre- and post-bariatric surgery [early (2 weeks) and late (12 months)] in patients with active idiopathic intracranial hypertension. Dynamic changes in gut neuropeptides (glucagon-like peptide-1, gastric inhibitory polypeptide and ghrelin) and metabolites (untargeted ultra-high performance liquid chromatography-mass spectrometry) were evaluated. We determined the relationship between gut neuropeptides, metabolites and intracranial pressure. Eighteen idiopathic intracranial hypertension patients were included [Roux-en-Y gastric bypass (RYGB) n = 7, gastric banding n = 6 or sleeve gastrectomy n = 5]. At 2 weeks post-bariatric surgery, despite similar weight loss, RYGB had a 2-fold (50%) greater reduction in intracranial pressure compared to sleeve. Increased meal-stimulated glucagon-like peptide-1 secretion was observed after RYGB (+600%) compared to sleeve (+319%). There was no change in gastric inhibitory polypeptide and ghrelin. Dynamic changes in meal-stimulated metabolites after bariatric surgery consistently identified changes in lipid metabolites, predominantly ceramides, glycerophospholipids and lysoglycerophospholipids, which correlated with intracranial pressure. A greater number of differential lipid metabolites were observed in the RYGB cohort at 2 weeks, and these also correlated with intracranial pressure. In idiopathic intracranial hypertension, we identified novel changes in lipid metabolites and meal-stimulated glucagon-like peptide-1 levels following bariatric surgery which were associated with changes in intracranial pressure. RYGB was most effective at reducing intracranial pressure despite analogous weight loss to gastric sleeve at 2 weeks post-surgery and was associated with more pronounced changes in these metabolite pathways. We suggest that these novel perturbations in lipid metabolism and glucagon-like peptide-1 secretion are mechanistically important in driving a reduction in intracranial pressure following weight loss in patients with idiopathic intracranial hypertension. Therapeutic targeting of these pathways, for example with glucagon-like peptide-1 agonist infusion, could represent a therapeutic strategy.
RESUMO
Untargeted metabolomics is an established approach in toxicology for characterising endogenous metabolic responses to xenobiotic exposure. Detecting the xenobiotic and its biotransformation products as part of the metabolomics analysis provides an opportunity to simultaneously gain deep insights into its fate and metabolism, and to associate the internal relative dose directly with endogenous metabolic responses. This integration of untargeted exposure and response measurements into a single assay has yet to be fully demonstrated. Here we assemble a workflow to discover and analyse pharmaceutical-related measurements from routine untargeted UHPLC-MS metabolomics datasets, derived from in vivo (rat plasma and cardiac tissue, and human plasma) and in vitro (human cardiomyocytes) studies that were principally designed to investigate endogenous metabolic responses to drug exposure. Our findings clearly demonstrate how untargeted metabolomics can discover extensive biotransformation maps, temporally-changing relative systemic exposure, and direct associations of endogenous biochemical responses to the internal dose.
Assuntos
Metabolômica , Xenobióticos , Humanos , Ratos , Animais , Metaboloma , BiotransformaçãoRESUMO
Biomarker discovery using biobank samples collected from veterinary clinics would deliver insights into the diverse population of pets and accelerate diagnostic development. The acquisition, preparation, processing, and storage of biofluid samples in sufficient volumes and at a quality suitable for later analysis with most suitable discovery methods remain challenging. Metabolomics analysis is a valuable approach to detect health/disease phenotypes. Pre-processing changes during preparation of plasma/serum samples may induce variability that may be overcome using dried blood spots (DBSs). We report a proof of principle study by metabolite fingerprinting applying UHPLC-MS of plasma and DBSs acquired from healthy adult dogs and cats (age range 1-9 years), representing each of 4 dog breeds (Labrador retriever, Beagle, Petit Basset Griffon Vendeen, and Norfolk terrier) and the British domestic shorthair cat (n = 10 per group). Blood samples (20 and 40 µL) for DBSs were loaded onto filter paper, air-dried at room temperature (3 h), and sealed and stored (4°C for ~72 h) prior to storage at -80°C. Plasma from the same blood draw (250 µL) was prepared and stored at -80°C within 1 h of sampling. Metabolite fingerprinting of the DBSs and plasma produced similar numbers of metabolite features that had similar abilities to discriminate between biological classes and correctly assign blinded samples. These provide evidence that DBSs, sampled in a manner amenable to application in in-clinic/in-field processing, are a suitable sample for biomarker discovery using UHPLC-MS metabolomics. Further, given appropriate owner consent, the volumes tested (20-40 µL) make the acquisition of remnant blood from blood samples drawn for other reasons available for biobanking and other research activities. Together, this makes possible large-scale biobanking of veterinary samples, gaining sufficient material sooner and enabling quicker identification of biomarkers of interest.
RESUMO
Dysregulated cellular metabolism is a cancer hallmark for which few druggable oncoprotein targets have been identified. Increased fatty acid (FA) acquisition allows cancer cells to meet their heightened membrane biogenesis, bioenergy, and signaling needs. Excess FAs are toxic to non-transformed cells but surprisingly not to cancer cells. Molecules underlying this cancer adaptation may provide alternative drug targets. Here, we demonstrate that diacylglycerol O-acyltransferase 1 (DGAT1), an enzyme integral to triacylglyceride synthesis and lipid droplet formation, is frequently up-regulated in melanoma, allowing melanoma cells to tolerate excess FA. DGAT1 over-expression alone transforms p53-mutant zebrafish melanocytes and co-operates with oncogenic BRAF or NRAS for more rapid melanoma formation. Antagonism of DGAT1 induces oxidative stress in melanoma cells, which adapt by up-regulating cellular reactive oxygen species defenses. We show that inhibiting both DGAT1 and superoxide dismutase 1 profoundly suppress tumor growth through eliciting intolerable oxidative stress.
Assuntos
Diacilglicerol O-Aciltransferase , Melanoma , Animais , Diacilglicerol O-Aciltransferase/genética , Diacilglicerol O-Aciltransferase/metabolismo , Proteínas Oncogênicas/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio , Triglicerídeos , Peixe-Zebra/metabolismoRESUMO
Currently there is limited information available on the accuracy and precision of relative isotopic abundance (RIA) measurements using high-resolution direct-infusion mass spectrometry (HR DIMS), and it is unclear if this information can benefit automated peak annotation in metabolomics. Here we characterize the accuracy of RIA measurements on the Thermo LTQ FT Ultra (resolution of 100,000-750,000) and LTQ Orbitrap (R = 100,000) mass spectrometers. This first involved reoptimizing the SIM-stitching method (Southam, A. D. Anal. Chem. 2007, 79, 4595-4602) for the LTQ FT Ultra, which achieved a ca. 3-fold sensitivity increase compared to the original method while maintaining a root-mean-squared mass error of 0.16 ppm. Using this method, we show the quality of RIA measurements is highly dependent on signal-to-noise ratio (SNR), with RIA accuracy increasing with higher SNR. Furthermore, a negative offset between the theoretical and empirically calculated numbers of carbon atoms was observed for both mass spectrometers. Increasing the resolution of the LTQ FT Ultra lowered both the sensitivity and the quality of RIA measurements. Overall, although the errors in the empirically calculated number of carbons can be large (e.g., 10 carbons), we demonstrate that RIA measurements do improve automated peak annotation, increasing the number of single empirical formula assignments by >3-fold compared to using accurate mass alone.
Assuntos
Espectrometria de Massas/métodos , Metaboloma , Isótopos de Carbono/química , Análise de Fourier , Espectrometria de Massas/instrumentaçãoRESUMO
The ability of targeted and nontargeted metabolomics to discover chronic ecotoxicological effects is largely unexplored. Fenitrothion, an organophosphate pesticide, is categorized as a "red list" pollutant, being particularly hazardous to aquatic life. It acts primarily as a cholinesterase inhibitor, but evidence suggests it can also act as an androgen receptor antagonist. Whole-organism fenitrothion-induced toxicity is well-established, but information regarding target and off-target molecular toxicities is limited. Here we study the molecular responses of male roach ( Rutilus rutilus ) exposed to fenitrothion, including environmentally realistic concentrations, for 28 days. Acetylcholine was assessed in brain; steroid metabolism was measured in testes and plasma; and NMR and mass spectrometry-based metabolomics were conducted on testes and liver to discover off-target toxicity. O-demethylation was confirmed as a major route of pesticide degradation. Fenitrothion significantly depleted acetylcholine, confirming its primary mode of action, and 11-ketotestosterone in plasma and cortisone in testes, showing disruption of steroid metabolism. Metabolomics revealed significant perturbations to the hepatic phosphagen system and previously undocumented effects on phenylalanine metabolism in liver and testes. On the basis of several unexpected molecular responses that were opposite to the anticipated acute toxicity, we propose that chronic pesticide exposure induces an adapting phenotype in roach, which may have considerable implications for interpreting molecular biomarker responses in field-sampled fish.
Assuntos
Cyprinidae/metabolismo , Fenitrotion/toxicidade , Inseticidas/toxicidade , Metaboloma/efeitos dos fármacos , Compostos Organofosforados/toxicidade , Poluentes Químicos da Água/toxicidade , Acetilcolina/metabolismo , Animais , Relação Dose-Resposta a Droga , Monitoramento Ambiental/métodos , Fenitrotion/metabolismo , Água Doce/química , Inseticidas/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Compostos Organofosforados/metabolismo , Testículo/efeitos dos fármacos , Testículo/metabolismo , Testosterona/análogos & derivados , Testosterona/metabolismo , Poluentes Químicos da Água/metabolismoRESUMO
Cytoglobin is important in the progression of oral squamous cell carcinoma but the molecular and cellular basis remain to be elucidated. In the current study, we develop a new cell model to study the function of cytoglobin in oral squamous carcinoma and response to cisplatin. Transcriptomic profiling showed cytoglobin mediated changes in expression of genes related to stress response, redox metabolism, mitochondrial function, cell adhesion, and fatty acid metabolism. Cellular and biochemical studies show that cytoglobin expression results in changes to phenotype associated with cancer progression including: increased cellular proliferation, motility and cell cycle progression. Cytoglobin also protects cells from cisplatin-induced apoptosis and oxidative stress with levels of the antioxidant glutathione increased and total and mitochondrial reactive oxygen species levels reduced. The mechanism of cisplatin resistance involved inhibition of caspase 9 activation and cytoglobin protected mitochondria from oxidative stress-induced fission. To understand the mechanism behind these phenotypic changes we employed lipidomic analysis and demonstrate that levels of the redox sensitive and apoptosis regulating cardiolipin are significantly up-regulated in cells expressing cytoglobin. In conclusion, our data shows that cytoglobin expression results in important phenotypic changes that could be exploited by cancer cells in vivo to facilitate disease progression.
Assuntos
Apoptose/efeitos dos fármacos , Cardiolipinas/metabolismo , Citoglobina/farmacologia , Mitocôndrias/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Antioxidantes/metabolismo , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/metabolismo , Adesão Celular/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Progressão da Doença , Glutationa/metabolismo , Humanos , Mitocôndrias/metabolismo , Neoplasias Bucais/tratamento farmacológico , Neoplasias Bucais/metabolismo , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transcriptoma/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
In the fight against antibiotic resistance, drugs that target resistance mechanisms in bacteria can be used to restore the therapeutic effectiveness of antibiotics. The multidrug resistance efflux complex AcrAB-TolC is the most clinically relevant efflux pump in Enterobacterales and is a target for drug discovery. Inhibition of the pump protein AcrB allows the intracellular accumulation of a wide variety of antibiotics, effectively restoring their therapeutic potency. To facilitate the development of AcrB efflux inhibitors, it is desirable to discover the native substrates of the pump, as these could be chemically modified to become inhibitors. We analyzed the native substrate profile of AcrB in Escherichia coli MG1655 and Salmonella enterica serovar Typhimurium SL1344 using an untargeted metabolomics approach. We analyzed the endo- and exometabolome of the wild-type strain and their respective AcrB loss-of-function mutants (AcrB D408A) to determine the metabolites that are native substrates of AcrB. Although there is 95% homology between the AcrB proteins of S. Typhimurium and E. coli, we observed mostly different metabolic responses in the exometabolomes of the S. Typhimurium and E. coli AcrB D408A mutants relative to those in the wild type, potentially indicating a differential metabolic adaptation to the same mutation in these two species. Additionally, we uncovered metabolite classes that could be involved in virulence of S. Typhimurium and a potential natural substrate of AcrB common to both species.IMPORTANCE Multidrug-resistant Gram-negative bacteria pose a global threat to human health. The AcrB efflux pump confers inherent and evolved drug resistance to Enterobacterales, including Escherichia coli and Salmonella enterica serovar Typhimurium. We provide insights into the physiological role of AcrB: (i) we observe that loss of AcrB function in two highly related species, E. coli and S. Typhimurium, has different biological effects despite AcrB conferring drug resistance to the same groups of antibiotics in both species, and (ii) we identify potential natural substrates of AcrB, some of which are in metabolite classes implicated in the virulence of S. Typhimurium. Molecules that inhibit multidrug efflux potentiate the activity of old, licensed, and new antibiotics. The additional significance of our research is in providing data about the identity of potential natural substrates of AcrB in both species. Data on these will facilitate the discovery of, and/or could be chemically modified to become, new efflux inhibitors.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Salmonella typhimurium/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Metabolômica , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genéticaRESUMO
Drug repurposing is a cost-effective means of targeting new therapies for cancer. We have examined the effects of the repurposed drugs, bezafibrate, medroxyprogesterone acetate and valproic acid on human osteosarcoma cells, i.e., SAOS2 and MG63 compared with their normal cell counterparts, i.e. mesenchymal stem/stromal cells (MSCs). Cell growth, viability and migration were measured by biochemical assay and live cell imaging, whilst levels of lipid-synthesising enzymes were measured by immunoblotting cell extracts. These drug treatments inhibited the growth and survival of SAOS2 and MG63 cells most effectively when used in combination (termed V-BAP). In contrast, V-BAP treated MSCs remained viable with only moderately reduced cell proliferation. V-BAP treatment also inhibited migratory cell phenotypes. MG63 and SAOS2 cells expressed much greater levels of fatty acid synthase and stearoyl CoA desaturase 1 than MSCs, but these elevated enzyme levels significantly decreased in the V-BAP treated osteosarcoma cells prior to cell death. Hence, we have identified a repurposed drug combination that selectively inhibits the growth and survival of human osteosarcoma cells in association with altered lipid metabolism without adversely affecting their non-transformed cell counterparts.
Assuntos
Bezafibrato/administração & dosagem , Neoplasias Ósseas/patologia , Proliferação de Células/efeitos dos fármacos , Acetato de Medroxiprogesterona/administração & dosagem , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteossarcoma/patologia , Ácido Valproico/administração & dosagem , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/enzimologia , Linhagem Celular Tumoral , Regulação para Baixo , Reposicionamento de Medicamentos , Quimioterapia Combinada , Ácido Graxo Sintases/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Osteossarcoma/tratamento farmacológico , Osteossarcoma/enzimologia , Estearoil-CoA Dessaturase/metabolismo , Regulação para CimaRESUMO
At selected sites around the UK, the offshore sentinel flatfish species dab Limanda limanda are found to contain elevated levels of macroscopic liver tumors. Previous proteomic and metabolomic studies have demonstrated that differences exist between tumor and non-tumor tissues; however, these differing features were not identified, and little is known about the changes at the gene expression level, or whether prognostic markers are present and can be identified. A flounder Platichthys flesus custom cDNA microarray and RT-PCR were used to investigate hepatic mRNA expression in the histologically confirmed tumors, hepatocellular adenoma (HA) and hepatocellular carcinoma (HC) from dab, and in adjacent normal tissue from the same fish. Differences in gene expression were observed between tumor and normal tissues, and between tumor types. A class-prediction approach using 50 transcripts revealed sufficient group-specific expression profiles to allow segregation of samples dependent on their tumor type or the sex of the host. Vitellogenins were found to display the greatest induction (up to 500-fold induction) in some HC tumors from female fish and in both HA and HC tumors from males. To the best of our knowledge, this is the first report of the association of vitellogenin expression with tumors of wild fish.
Assuntos
Adenoma de Células Hepáticas/veterinária , Carcinoma Hepatocelular/veterinária , Doenças dos Peixes/metabolismo , Linguados/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Neoplasias Hepáticas/veterinária , Adenoma de Células Hepáticas/metabolismo , Animais , Carcinoma Hepatocelular/metabolismo , Feminino , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Perfilação da Expressão Gênica , Fígado/patologia , Neoplasias Hepáticas/metabolismo , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Lipids play a significant role in regulation of health and disease. To enhance our understanding of the role of lipids in regulation of lifespan and healthspan additional studies are required. Here, UHPLC-MS/MS lipidomics was used to measure dynamic changes in lipid composition as a function of age and gender in genetically identical male and female Daphnia magna with different average lifespans. We demonstrate statistically significant age-related changes in triglycerides (TG), diglycerides (DG), phosphatidylcholine, phosphatidylethanolamine, ceramide and sphingomyelin lipid groups, for example, in males, 17.04% of TG lipid species decline with age whilst 37.86% increase in relative intensity with age. In females, 23.16% decrease and 25.31% increase in relative intensity with age. Most interestingly, the rate and direction of change can differ between genetically identical female and male Daphnia magna, which could be the cause and/or the consequence of the different average lifespans between the two genetically identical genders. This study provides a benchmark dataset to understand how lipids alter as a function of age in genetically identical female and male species with different average lifespan and ageing rate.