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Children are susceptible to influenza infections and can experience severe disease presentation due to a lack of or limited pre-existing immunity. Despite the disproportionate impact influenza has on this population, there is a lack of focus on pediatric influenza research, particularly when it comes to identifying the pathogenesis of long-term outcomes that persist beyond the point of viral clearance. In this study, juvenile outbred male and female mice were infected with influenza and analyzed following viral clearance to determine how sex impacts the persistent inflammatory responses to influenza. It was found that females maintained a broader cytokine response in the lung following clearance of influenza, with innate, type I and type II cytokine signatures in almost all mice. Males, on the other hand, had higher levels of IL-6 and other macrophage-related cytokines, but no evidence of a type I or type II response. The immune landscape was similar in the lungs between males and females postinfection, but males had a higher regulatory T cell to TH1 ratio compared with female mice. Cytokine production positively correlated with the frequency of TH1 cells and exudate macrophages, as well as the number of cells in the bronchoalveolar lavage fluid. Furthermore, female lungs were enriched for metabolites involved in the glycolytic pathway, suggesting glycolysis is higher in female lungs compared with males after viral clearance. These data suggest juvenile female mice have persistent and excessive lung inflammation beyond the point of viral clearance, whereas juvenile males had a more immunosuppressive phenotype.NEW & NOTEWORTHY This study identifies sex-based differences in persistent lung inflammation following influenza infection in an outbred, juvenile animal model of pediatric infection. These findings indicate the importance of considering sex and age as variable in infectious disease research.
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Citocinas , Infecciones por Orthomyxoviridae , Neumonía , Caracteres Sexuales , Animales , Femenino , Masculino , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/virología , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/metabolismo , Ratones , Citocinas/metabolismo , Neumonía/virología , Neumonía/patología , Neumonía/inmunología , Neumonía/metabolismo , Pulmón/virología , Pulmón/patología , Pulmón/inmunología , Pulmón/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/patología , Macrófagos/virología , Factores SexualesRESUMEN
The monomeric heme protein myoglobin (Mb), traditionally thought to be expressed exclusively in cardiac and skeletal muscle, is now known to be expressed in approximately 40% of breast tumors. While Mb expression is associated with better patient prognosis, the molecular mechanisms by which Mb limits cancer progression are unclear. In muscle, Mb's predominant function is oxygen storage and delivery, which is dependent on the protein's heme moiety. However, prior studies demonstrate that the low levels of Mb expressed in cancer cells preclude this function. Recent studies propose a novel fatty acid binding function for Mb via a lysine residue (K46) in the heme pocket. Given that cancer cells can upregulate fatty acid oxidation (FAO) to maintain energy production for cytoskeletal remodeling during cell migration, we tested whether Mb-mediated fatty acid binding modulates FAO to decrease breast cancer cell migration. We demonstrate that the stable expression of human Mb in MDA-MB-231 breast cancer cells decreases cell migration and FAO. Site-directed mutagenesis of Mb to disrupt Mb fatty acid binding did not reverse Mb-mediated attenuation of FAO or cell migration in these cells. In contrast, cells expressing Apo-Mb, in which heme incorporation was disrupted, showed a reversal of Mb-mediated attenuation of FAO and cell migration, suggesting that Mb attenuates FAO and migration via a heme-dependent mechanism rather than through fatty acid binding. To this end, we show that Mb's heme-dependent oxidant generation propagates dysregulated gene expression of migratory genes, and this is reversed by catalase treatment. Collectively, these data demonstrate that Mb decreases breast cancer cell migration, and this effect is due to heme-mediated oxidant production rather than fatty acid binding. The implication of these results will be discussed in the context of therapeutic strategies to modulate oxidant production and Mb in tumors. Highlights: Myoglobin (Mb) expression in MDA-MB-231 breast cancer cells slows migration.Mb expression decreases mitochondrial respiration and fatty acid oxidation.Mb-dependent fatty acid binding does not regulate cell migration or respiration.Mb-dependent oxidant generation decreases mitochondrial metabolism and migration.Mb-derived oxidants dysregulate migratory gene expression.
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Dicarboxylic fatty acids are generated in the liver and kidney in a minor pathway called fatty acid ω-oxidation. The effects of consuming dicarboxylic fatty acids as an alternative source of dietary fat have not been explored. Here, we fed dodecanedioic acid, a 12-carbon dicarboxylic (DC12), to mice at 20% of daily caloric intake for nine weeks. DC12 increased metabolic rate, reduced body fat, reduced liver fat, and improved glucose tolerance. We observed DC12-specific breakdown products in liver, kidney, muscle, heart, and brain, indicating that oral DC12 escaped first-pass liver metabolism and was utilized by many tissues. In tissues expressing the "a" isoform of acyl-CoA oxidase-1 (ACOX1), a key peroxisomal fatty acid oxidation enzyme, DC12 was chain shortened to the TCA cycle intermediate succinyl-CoA. In tissues with low peroxisomal fatty acid oxidation capacity, DC12 was oxidized by mitochondria. In vitro, DC12 was catabolized even by adipose tissue and was not stored intracellularly. We conclude that DC12 and other dicarboxylic acids may be useful for combatting obesity and for treating metabolic disorders.
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The enzymatic oxidation of arachidonic acid is proposed to yield trihydroxytetraene species (termed lipoxins) that resolve inflammation via ligand activation of the formyl peptide receptor, FPR2. While cell and murine models activate signaling responses to synthetic lipoxins, primarily 5S,6R,15S-trihydroxy-7E,9E,11Z,13E-eicosatetraenoic acid (lipoxin A4, LXA4), there are expanding concerns about the biological formation, detection and signaling mechanisms ascribed to LXA4 and related di- and tri-hydroxy ω-6 and ω-3 fatty acids. Herein, the generation and actions of LXA4 and its primary 15-oxo metabolite were assessed in control, LPS-activated and arachidonic acid supplemented RAW 264.7 macrophages. Despite protein expression of all enzymes required for LXA4 synthesis, both LXA4 and its 15-oxo-LXA4 metabolite were undetectable. Moreover, synthetic LXA4 and the membrane permeable 15-oxo-LXA4 methyl ester that is rapidly de-esterified to 15-oxo-LXA4, displayed no ligand activity for the putative LXA4 receptor FPR2, as opposed to the FPR2 ligand WKYMVm. Alternatively, 15-oxo-LXA4, an electrophilic α,ß-unsaturated ketone, alkylates nucleophilic amino acids such as cysteine to modulate redox-sensitive transcriptional regulatory protein and enzyme function. 15-oxo-LXA4 activated nuclear factor (erythroid related factor 2)-like 2 (Nrf2)-regulated gene expression of anti-inflammatory and repair genes and inhibited nuclear factor (NF)-κB-regulated pro-inflammatory mediator expression. LXA4 did not impact these macrophage anti-inflammatory and repair responses. In summary, these data show an absence of macrophage LXA4 formation and receptor-mediated signaling actions. Rather, if LXA4 were present in sufficient concentrations, this, and other more abundant mono- and poly-hydroxylated unsaturated fatty acids can be readily oxidized to electrophilic α,ß-unsaturated ketone products that modulate the redox-sensitive cysteine proteome via G-protein coupled receptor-independent mechanisms.
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Aberrant mitochondrial fission/fusion dynamics have been reported in cancer cells. While post translational modifications are known regulators of the mitochondrial fission/fusion machinery, we show that alternative splice variants of the fission protein Drp1 (DNM1L) have specific and unique roles in cancer, adding to the complexity of mitochondrial fission/fusion regulation in tumor cells. Ovarian cancer specimens express an alternative splice transcript variant of Drp1 lacking exon 16 of the variable domain, and high expression of this splice variant relative to other transcripts is associated with poor patient outcome. Unlike the full-length variant, expression of Drp1 lacking exon 16 leads to decreased association of Drp1 to mitochondrial fission sites, more fused mitochondrial networks, enhanced respiration, and TCA cycle metabolites, and is associated with a more metastatic phenotype in vitro and in vivo. These pro-tumorigenic effects can also be inhibited by specific siRNA-mediated inhibition of the endogenously expressed transcript lacking exon 16. Moreover, lack of exon 16 abrogates mitochondrial fission in response to pro-apoptotic stimuli and leads to decreased sensitivity to chemotherapeutics. These data emphasize the significance of the pathophysiological consequences of Drp1 alternative splicing and divergent functions of Drp1 splice variants, and strongly warrant consideration of Drp1 splicing in future studies.
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Metabolic reprogramming in pediatric diffuse midline glioma is driven by gene expression changes induced by the hallmark histone mutation H3K27M, which results in aberrantly permissive activation of oncogenic signaling pathways. Previous studies of diffuse midline glioma with altered H3K27 (DMG-H3K27a) have shown that the RAS pathway, specifically through its downstream kinase, extracellular-signal-related kinase 5 (ERK5), is critical for tumor growth. Further downstream effectors of ERK5 and their role in DMG-H3K27a metabolic reprogramming have not been explored. We establish that ERK5 is a critical regulator of cell proliferation and glycolysis in DMG-H3K27a. We demonstrate that ERK5 mediates glycolysis through activation of transcription factor MEF2A, which subsequently modulates expression of glycolytic enzyme PFKFB3. We show that in vitro and mouse models of DMG-H3K27a are sensitive to the loss of PFKFB3. Multi-targeted drug therapy against the ERK5-PFKFB3 axis, such as with small-molecule inhibitors, may represent a promising therapeutic approach in patients with pediatric diffuse midline glioma.
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Glioma , Histonas , Animales , Niño , Humanos , Ratones , Quinasas MAP Reguladas por Señal Extracelular , Glioma/genética , Glucólisis , Histonas/genética , Fosfofructoquinasa-2 , Monoéster Fosfórico Hidrolasas , Transducción de SeñalRESUMEN
DNA damage and cellular metabolism are intricately linked with bidirectional feedback. Two of the main effectors of the DNA damage response and control of cellular metabolism are ATR and mTORC1, respectively. Prior work has placed ATR upstream of mTORC1 during replication stress, yet the direct mechanism for how mTORC1 is activated in this context remain unclear. We previously published that p16-low cells have mTORC1 hyperactivation, which in part promotes their proliferation. Using this model, we found that ATR, but not ATM, is upstream of mTORC1 activation via de novo cholesterol synthesis and is associated with increased lanosterol synthase (LSS). Indeed, p16-low cells showed increased cholesterol abundance. Additionally, knockdown of either ATR or LSS decreased mTORC1 activity. Decreased mTORC1 activity due to ATR knockdown was rescued by cholesterol supplementation. Finally, using both LSS inhibitors and multiple FDA-approved de novo cholesterol synthesis inhibitors, we found that the de novo cholesterol biosynthesis pathway is a metabolic vulnerability of p16-low cells. Together, our data provide new evidence coupling the DNA damage response and cholesterol metabolism and demonstrate the feasibility of using FDA-approved cholesterol-lowering drugs in tumors with loss of p16.
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Loss of oral tolerance (LOT) to gluten, driven by dendritic cell (DC) priming of gluten-specific T helper 1 (Th1) cell immune responses, is a hallmark of celiac disease (CeD) and can be triggered by enteric viral infections. Whether certain commensals can moderate virus-mediated LOT remains elusive. Here, using a mouse model of virus-mediated LOT, we discovered that the gut-colonizing protist Tritrichomonas (T.) arnold promotes oral tolerance and protects against reovirus- and murine norovirus-mediated LOT, independent of the microbiota. Protection was not attributable to antiviral host responses or T. arnold-mediated innate type 2 immunity. Mechanistically, T. arnold directly restrained the proinflammatory program in dietary antigen-presenting DCs, subsequently limiting Th1 and promoting regulatory T cell responses. Finally, analysis of fecal microbiomes showed that T. arnold-related Parabasalid strains are underrepresented in human CeD patients. Altogether, these findings will motivate further exploration of oral-tolerance-promoting protists in CeD and other immune-mediated food sensitivities.
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Antígenos , Inmunidad Innata , Animales , Ratones , Humanos , Dieta , Glútenes , Células Dendríticas , Tolerancia InmunológicaRESUMEN
PURPOSE: Diastolic dysfunction is an increasingly common cardiac pathology linked to heart failure with preserved ejection fraction. Previous studies have implicated glucagon-like peptide 1 (GLP-1) receptor agonists as potential therapies for improving diastolic dysfunction. In this study, we investigate the physiologic and metabolic changes in a mouse model of angiotensin II (AngII)-mediated diastolic dysfunction with and without the GLP-1 receptor agonist liraglutide (Lira). METHODS: Mice were divided into sham, AngII, or AngII+Lira therapy for 4 weeks. Mice were monitored for cardiac function, weight change, and blood pressure at baseline and after 4 weeks of treatment. After 4 weeks of treatment, tissue was collected for histology, protein analysis, targeted metabolomics, and protein synthesis assays. RESULTS: AngII treatment causes diastolic dysfunction when compared to sham mice. Lira partially prevents this dysfunction. The improvement in function in Lira mice is associated with dramatic changes in amino acid accumulation in the heart. Lira mice also have improved markers of protein translation by Western blot and increased protein synthesis by puromycin assay, suggesting that increased protein turnover protects against fibrotic remodeling and diastolic dysfunction seen in the AngII cohort. Lira mice also lost lean muscle mass compared to the AngII cohort, raising concerns about peripheral muscle scavenging as a source of the increased amino acids in the heart. CONCLUSIONS: Lira therapy protects against AngII-mediated diastolic dysfunction, at least in part by promoting amino acid uptake and protein turnover in the heart. Liraglutide therapy is associated with loss of mean muscle mass, and long-term studies are warranted to investigate sarcopenia and frailty with liraglutide therapy in the setting of diastolic disease.
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Obesity and associated changes to the gut microbiome worsen airway inflammation and hyperresponsiveness in asthma. Obesogenic host-microbial metabolomes have altered production of metabolites that may influence lung function and inflammatory responses in asthma. To understand the interplay of the gut microbiome, metabolism, and host inflammation in obesity-associated asthma, we used a multi-omics approach to profile the gut-lung axis in the setting of allergic airway disease and diet-induced obesity. We evaluated an immunomodulator, nitro-oleic acid (NO2-OA), as a host- and microbial-targeted treatment intervention for obesity-associated allergic asthma. Allergic airway disease was induced using house dust mite and cholera toxin adjuvant in C57BL6/J mice with diet-induced obesity to model obesity-associated asthma. Lung function was measured by flexiVent following a week of NO2-OA treatment and allergen challenge. 16S rRNA gene (from DNA, taxa presence) and 16S rRNA (from RNA, taxa activity) sequencing, metabolomics, and host gene expression were paired with a Treatment-Measured-Response model as a data integration framework for identifying latent/hidden relationships with linear regression among variables identified from high-dimensional meta-omics datasets. Targeting both the host and gut microbiota, NO2-OA attenuated airway inflammation, improved lung elastance, and modified the gut microbiome. Meta-omics data integration and modeling determined that gut-associated inflammation, metabolites, and functionally active gut microbiota were linked to lung function outcomes. Using Treatment-Measured-Response modeling and meta-omics profiling of the gut-lung axis, we uncovered a previously hidden network of interactions between gut levels of amino acid metabolites involved in elastin and collagen synthesis, gut microbiota, NO2-OA, and lung elastance. Further targeted metabolomics analyses revealed that obese mice with allergic airway disease had higher levels of proline and hydroxyproline in the lungs. NO2-OA treatment reduced proline biosynthesis by downregulation of pyrroline-5-carboxylate reductase 1 (PYCR1) expression. These findings are relevant to human disease: adults with mild-moderate asthma and BMI ≥ 25 had higher plasma hydroxyproline levels. Our results suggest that changes to structural proteins in the lung airways and parenchyma may contribute to heightened lung elastance and serve as a potential therapeutic target for obese allergic asthma.
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The use of probiotics by cancer patients is increasing, including among those undergoing immune checkpoint inhibitor (ICI) treatment. Here, we elucidate a critical microbial-host crosstalk between probiotic-released aryl hydrocarbon receptor (AhR) agonist indole-3-aldehyde (I3A) and CD8 T cells within the tumor microenvironment that potently enhances antitumor immunity and facilitates ICI in preclinical melanoma. Our study reveals that probiotic Lactobacillus reuteri (Lr) translocates to, colonizes, and persists within melanoma, where via its released dietary tryptophan catabolite I3A, it locally promotes interferon-γ-producing CD8 T cells, thereby bolstering ICI. Moreover, Lr-secreted I3A was both necessary and sufficient to drive antitumor immunity, and loss of AhR signaling within CD8 T cells abrogated Lr's antitumor effects. Further, a tryptophan-enriched diet potentiated both Lr- and ICI-induced antitumor immunity, dependent on CD8 T cell AhR signaling. Finally, we provide evidence for a potential role of I3A in promoting ICI efficacy and survival in advanced melanoma patients.
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Limosilactobacillus reuteri , Melanoma , Microambiente Tumoral , Humanos , Dieta , Inhibidores de Puntos de Control Inmunológico , Limosilactobacillus reuteri/metabolismo , Melanoma/terapia , Triptófano/metabolismo , Linfocitos T CD8-positivos/inmunología , Receptores de Hidrocarburo de Aril/agonistasRESUMEN
BACKGROUND: Sepsis is a common and deadly syndrome, accounting for more than 11 million deaths annually. To mature a deeper understanding of the host and pathogen mechanisms contributing to poor outcomes in sepsis, and thereby possibly inform new therapeutic targets, sophisticated, and expensive biorepositories are typically required. We propose that remnant biospecimens are an alternative for mechanistic sepsis research, although the viability and scientific value of such remnants are unknown. METHODS AND RESULTS: The Remnant Biospecimen Investigation in Sepsis study is a prospective cohort study of 225 adults (age ≥ 18 yr) presenting to the emergency department with community sepsis, defined as sepsis-3 criteria within 6 hours of arrival. The primary objective was to determine the scientific value of a remnant biospecimen repository in sepsis linked to clinical phenotyping in the electronic health record. We will study candidate multiomic readouts of sepsis biology, governed by a conceptual model, and determine the precision, accuracy, integrity, and comparability of proteins, small molecules, lipids, and pathogen sequencing in remnant biospecimens compared with paired biospecimens obtained according to research protocols. Paired biospecimens will include plasma from sodium-heparin, EDTA, sodium fluoride, and citrate tubes. CONCLUSIONS: The study has received approval from the University of Pittsburgh Human Research Protection Office (Study 21120013). Recruitment began on October 25, 2022, with planned release of primary results anticipated in 2024. Results will be made available to the public, the funders, critical care societies, laboratory medicine scientists, and other researchers.
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BACKGROUND: Bronchoalveolar lavage (BAL) fluid prostaglandin D2(PGD2) levels are increased in patients with severe, poorly controlled asthma in association with epithelial mast cells (MCs). PGD2, which is generated by hematopoietic prostaglandin D synthase (HPGDS), acts on 3 G protein-coupled receptors, including chemoattractant receptor-homologous molecule expressed on TH2 lymphocytes (CRTH2) and PGD2 receptor 1 (DP1). However, much remains to be understood regarding the presence and activation of these pathway elements in asthmatic patients. OBJECTIVE: We sought to compare the expression and activation of PGD2 pathway elements in bronchoscopically obtained samples from healthy control subjects and asthmatic patients across a range of disease severity and control, as well as in relation to TH2 pathway elements. METHODS: Epithelial cells and BAL fluid were evaluated for HPGDS (quantitative real-time PCR/immunohistochemistry [IHC]) and PGD2 (ELISA/liquid chromatography mass spectrometry) in relation to levels of MC proteases. Expression of the 2 inflammatory cell receptors DP1 and CRTH2 was evaluated on luminal cells. These PGD2 pathway markers were then compared with asthma severity, level of control, and markers of TH2 inflammation (blood eosinophils and fraction of exhaled nitric oxide). RESULTS: Confirming previous results, BAL fluid PGD2 levels were highest in patients with severe asthma (overall P = .0001). Epithelial cell compartment HPGDS mRNA and IHC values differed among groups (P = .008 and P < .0001, respectively) and correlated with MC protease mRNA. CRTH2 mRNA and IHC values were highest in patients with severe asthma (P = .001 and P = .0001, respectively). Asthma exacerbations, poor asthma control, and TH2 inflammatory markers were associated with higher PGD2, HPGDS, and CRTH2 levels. CONCLUSION: The current study identifies coordinated upregulation of the PGD2 pathway in patients with severe, poorly controlled, TH2-high asthma despite corticosteroid use.
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Asma/inmunología , Asma/metabolismo , Prostaglandina D2/metabolismo , Receptores Inmunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Transducción de Señal , Adulto , Asma/prevención & control , Líquido del Lavado Bronquioalveolar/inmunología , Estudios de Casos y Controles , Femenino , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Oxidorreductasas Intramoleculares/genética , Oxidorreductasas Intramoleculares/metabolismo , Lipocalinas/genética , Lipocalinas/metabolismo , Masculino , Persona de Mediana Edad , Receptores Inmunológicos/genética , Receptores de Prostaglandina/genética , Mucosa Respiratoria/metabolismo , Células Th2/inmunología , Células Th2/metabolismo , Regulación hacia Arriba , Adulto JovenRESUMEN
Nitrated fatty acids are the product of nitrogen dioxide reaction with unsaturated fatty acids. The discovery of peroxynitrite and peroxidase-induced nitration of biomolecules led to the initial reports of endogenous nitrated fatty acids. These species increase during ischemia/reperfusion, but concentrations are often at or near the limits of detection. Here, we describe multiple methods for nitrated fatty acid synthesis and sample extraction from complex biological matrices and a rigorous method of qualitative and quantitative detection of nitrated fatty acids by liquid chromatography-mass spectrometry. In addition, optimized instrument conditions and caveats regarding data interpretation are discussed.
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Ácidos Grasos/análisis , Ácidos Grasos/síntesis química , Nitratos/análisis , Nitratos/síntesis químicaRESUMEN
Multi-drug resistance protein (MRP) 4, an ATP-binding cassette (ABC) transporter, has broad substrate specificity. It facilitates the transport of bile salt conjugates, conjugated steroids, nucleoside analogs, eicosanoids, and cardiovascular drugs. Recent studies in liver carcinoma cells and hepatocytes showed that MRP4 expression is regulated by the aryl hydrocarbon receptor (AhR) and nuclear factor E2-related factor 2 (Nrf2). The AhR has particular importance in the lung and is most commonly associated with the up-regulation of cytochrome P-450 (CYP)-mediated metabolism of benzo[a]pyrene (B[a]P) to reactive intermediates. Treatment of H358, human bronchoalveolar, cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or (-)-benzo[a]pyrene-7,8-dihydro-7,8-diol (B[a]P-7,8-dihydrodiol), the proximate carcinogen of B[a]P, revealed that MRP4 expression was increased compared to control. This suggested that MRP4 expression might contribute to the paradoxical decrease in (+)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene-2'-deoxyguanosine ((+)-anti-trans-B[a]PDE-dGuo) DNA-adducts observed in TCDD-treated H358 cells. We have now found that decreased MRP4 expression induced by a short hairpin RNA (shRNA), or chemical inhibition with probenecid, increased (+)-anti-trans-B[a]PDE-dGuo formation in cells treated with (-)-B[a]P-7,8-dihydrodiol, but not the ultimate carcinogen (+)-anti-trans-B[a]PDE. Thus, up-regulation of MRP4 increased cellular efflux of (-)-B[a]P-7,8-dihydrodiol, which attenuated DNA-adduct formation. This is the first report identifying a specific MRP efflux transporter that decreases DNA damage arising from an environmental carcinogen.
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Benzo(a)pireno/antagonistas & inhibidores , Benzo(a)pireno/toxicidad , Bronquios/citología , Carcinógenos/antagonistas & inhibidores , Carcinógenos/toxicidad , Aductos de ADN/efectos de los fármacos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/farmacología , Alveolos Pulmonares/citología , Bronquios/efectos de los fármacos , Líquido del Lavado Bronquioalveolar/citología , Línea Celular , ADN/genética , ADN/aislamiento & purificación , Dinoprostona/biosíntesis , Humanos , Inmunohistoquímica , Factor 2 Relacionado con NF-E2/metabolismo , Dibenzodioxinas Policloradas/toxicidad , Probenecid/farmacología , Alveolos Pulmonares/efectos de los fármacos , ARN Interferente Pequeño/biosíntesis , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor Cross-Talk/efectos de los fármacos , Receptores de Hidrocarburo de Aril/efectos de los fármacos , Fármacos Renales/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers(1,2). These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer(3-7). Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)(1,8). Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products. While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis(9). After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity(10,11). Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids(12). This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites(13). Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.
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Cromatografía Liquida/métodos , Lípidos/química , Espectrometría de Masas en Tándem/métodos , Ácido Araquidónico/metabolismo , Medios de Cultivo , Medición de Intercambio de Deuterio/métodos , Ácido Linoleico/metabolismo , Metabolismo de los Lípidos , Lípidos/aislamiento & purificación , Especies Reactivas de Oxígeno/metabolismo , EstereoisomerismoRESUMEN
Stable isotope dilution mass spectrometry (MS) represents the gold standard for quantification of endogenously formed cellular metabolites. Although coenzyme A (CoA) and acyl-CoA thioester derivatives are central players in numerous metabolic pathways, the lack of a commercially available isotopically labeled CoA limits the development of rigorous MS-based methods. In this study, we adapted stable isotope labeling by amino acids in cell culture (SILAC) methodology to biosynthetically generate stable isotope labeled CoA and thioester analogues for use as internal standards in liquid chromatography/multiple reaction monitoring mass spectrometry (LC/MRM-MS) assays. This was accomplished by incubating murine hepatocytes (Hepa 1c1c7) in media in which pantothenate (a precursor of CoA) was replaced with [(13)C(3)(15)N(1)]-pantothenate. Efficient incorporation into various CoA species was optimized to >99% [(13)C(3)(15)N(1)]-pantothenate after three passages of the murine cells in culture. Charcoal-dextran-stripped fetal bovine serum (FBS) was found to be more efficient for serum supplementation than dialyzed or undialyzed FBS, due to lower contaminating unlabeled pantothenate content. Stable isotope labeled CoA species were extracted and utilized as internal standards for CoA thioester analysis in cell culture models. This methodology of stable isotope labeling by essential nutrients in cell culture (SILEC) can serve as a paradigm for using vitamins and other essential nutrients to generate stable isotope standards that cannot be readily synthesized.
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Coenzima A/química , Marcaje Isotópico/métodos , Ácido Pantoténico/química , Animales , Células Cultivadas , Cromatografía Liquida , Coenzima A/biosíntesis , Coenzima A/aislamiento & purificación , Ésteres , Hepatocitos/citología , Hepatocitos/metabolismo , Ratones , Reproducibilidad de los Resultados , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a carcinogenic nitrosamine produced upon curing tobacco. It is present in tobacco smoke and undergoes metabolism to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in the lungs. NNAL undergoes further uridine diphosphate glucuronosyltransferase (UGT)-mediated metabolism to give N- and O-glucuronide metabolites, which together with free (non-conjugated) NNAL are then excreted in the urine. The ability to conduct validated analyses of free and conjugated NNAL in human urine is important in order to assess inter-individual differences in lung cancer risk from exposure to cigarette smoke. The use of stable isotope dilution (SID) methodology in combination with liquid chromatography/multiple reaction monitoring/mass spectrometry (LC/MRM-MS) provides the highest bioanalytical specificity possible for such analyses. We describe a novel derivatization procedure, which results in the formation of a pre-ionized N-propyl-NNAL derivative. The increased LC/MS sensitivity arising from this derivative then makes it possible to analyze free NNAL in only 0.25 mL urine. This substantial reduction in urine volume when compared with other methods that have been developed will help preserve the limited amounts of stored urine samples that are available from on-going longitudinal biomarker studies. The new high sensitivity SID LC/MRM-MS assay was employed to determine free and conjugated NNAL concentrations in urine samples from 60 individual disease-free smokers. Effects of inter-individual differences in urinary creatinine clearance on NNAL concentrations were then assessed and three metabolizer phenotypes were identified in the 60 subjects from the ratio of urinary NNAL glucuronides/free NNAL. Poor metabolizers (PMs, 14 subjects) with a ratio of NNAL glucuronides/free NNAL <2 (mean = 1.3), intermediate metabolizers (IMs, 36 subjects) with a ratio between 2 and 5 (mean = 3.4), and extensive metabolizers (EMs, 10 subjects) with a ratio >5 (mean = 11.1).