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For either healthy or diseased organisms, lipids are key components for cellular membranes; they play important roles in numerous cellular processes including cell growth, proliferation, differentiation, energy storage and signaling. Exercise and disease development are examples of cellular environment alterations which produce changes in these networks. There are indications that alterations in lipid metabolism contribute to the development and progression of a variety of cancers. Measuring such alterations and understanding the pathways involved is critical to fully understand cellular metabolism. The demands for this information have led to the emergence of lipidomics, which enables the large-scale study of lipids using mass spectrometry (MS) techniques. Mass spectrometry has been widely used in lipidomics and allows us to analyze detailed lipid profiles of cancers. In this article, we discuss emerging strategies for lipidomics by mass spectrometry; targeted, as opposed to global, lipid analysis provides an exciting new alternative method. Additionally, we provide an introduction to lipidomics, lipid categories and their major biological functions, along with lipidomics studies by mass spectrometry in cancer samples. Further, we summarize the importance of lipid metabolism in oncology and tumor microenvironment, some of the challenges for lipodomics, and the potential for targeted approaches for screening pharmaceutical candidates to improve the therapeutic efficacy of treatment in cancer patients.
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Acute inflammation is the body's first defense in response to pathogens or injury that is partially governed by a novel genus of endogenous lipid mediators that orchestrate the resolution of inflammation, coined specialized pro-resolving mediators (SPMs). SPMs, derived from omega-3-polyunstaturated fatty acids (PUFAs), include the eicosapentaenoic acid-derived and docosahexaenoic acid-derived Resolvins, Protectins, and Maresins. Herein, we review their biosynthesis, structural characteristics, and therapeutic effectiveness in various diseases such as ischemia, viral infections, periodontitis, neuroinflammatory diseases, cystic fibrosis, lung inflammation, herpes virus, and cancer, especially focusing on therapeutic effectiveness in respiratory inflammation and ischemia-related injuries. Resolvins are sub-nanomolar potent agonists that accelerate the resolution of inflammation by reducing excessive neutrophil infiltration, stimulating macrophage functions including phagocytosis, efferocytosis, and tissue repair. In addition to regulating neutrophils and macrophages, Resolvins control dendritic cell migration and T cell responses, and they also reduce the pro-inflammatory cytokines, proliferation, and metastasis of cancer cells. Importantly, several lines of evidence have demonstrated that Resolvins reduce tumor progression in melanoma, oral squamous cell carcinoma, lung cancer, and liver cancer. In addition, Resolvins enhance tumor cell debris clearance by macrophages in the tumor's microenvironment. Resolvins, with their unique stereochemical structure, receptors, and biosynthetic pathways, provide a novel therapeutical approach to activating resolution mechanisms during cancer progression.
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T cells are often absent from human cancer tissues during both spontaneously induced immunity and therapeutic immunotherapy, even in the presence of a functional T cell-recruiting chemokine system, suggesting the existence of T cell exclusion mechanisms that impair infiltration. Using a genome-wide in vitro screening platform, we identified a role for phospholipase A2 group 10 (PLA2G10) protein in T cell exclusion. PLA2G10 up-regulation is widespread in human cancers and is associated with poor T cell infiltration in tumor tissues. PLA2G10 overexpression in immunogenic mouse tumors excluded T cells from infiltration, resulting in resistance to anti-PD-1 immunotherapy. PLA2G10 can hydrolyze phospholipids into small lipid metabolites, thus inhibiting chemokine-mediated T cell mobility. Ablation of PLA2G10's enzymatic activity enhanced T cell infiltration and sensitized PLA2G10-overexpressing tumors to immunotherapies. Our study implicates a role for PLA2G10 in T cell exclusion from tumors and suggests a potential target for cancer immunotherapy.
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Neoplasias , Linfocitos T , Regulación hacia Arriba , Animales , Femenino , Humanos , Ratones , Línea Celular Tumoral , Inmunoterapia/métodos , Linfocitos Infiltrantes de Tumor/inmunología , Ratones Endogámicos C57BL , Neoplasias/inmunología , Fosfolipasas A/inmunología , Fosfolipasas A/genética , Fosfolipasas A2/inmunología , Linfocitos T/inmunología , Regulación hacia Arriba/inmunologíaRESUMEN
BACKGROUND: Malignant meningiomas are fatal and lack effective therapy. As M2 macrophages are the most prevalent immune cell type in human meningiomas, we hypothesized that normalizing this immunosuppressive population would be an effective treatment strategy. METHODS: We used CIBERSORTx to examine the proportions of 22 immune subsets in human meningiomas. We targeted the colony-stimulating factor 1 (CSF1) or CSF1 receptor (CSF1R) axis, an important regulator of macrophage phenotype, using monoclonal antibodies (mAbs) in a novel immunocompetent murine model (MGS1) for malignant meningioma. RNA sequencing (RNA-seq) was performed to identify changes in gene expression in the tumor microenvironment (TME). Mass cytometry was used to delineate changes in immune subsets after treatment. We measured patients' plasma CSF1 levels using ELISA and CSF1R expression using multiplex quantitative immunofluorescence in a human meningioma tissue microarray. RESULTS: Human meningiomas are heavily enriched for immunosuppressive myeloid cells. MGS1 recapitulates the TME of human meningiomas, including an abundance of myeloid cells, a paucity of infiltrating T cells, and low programmed death ligand 1 (PD-L1) expression. Treatment of murine meningiomas with anti-CSF1/CSF1R, but not programmed cell death receptor 1 (PD-1), mAbs abrogate tumor growth. RNA-seq and mass cytometry analyses reveal a myeloid cell reprogramming with limited effect on T cells in the TME. CSF1 plasma levels are significantly elevated in human patients, and CSF1R is highly expressed on CD163+ macrophages within the human TME. CONCLUSION: Our findings suggest that anti-CSF1/CSF1R antibody treatment may be an effective normalization cancer immunotherapy for malignant meningiomas.
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Factor Estimulante de Colonias de Macrófagos , Neoplasias Meníngeas , Meningioma , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos , Animales , Humanos , Macrófagos , Neoplasias Meníngeas/tratamiento farmacológico , Meningioma/tratamiento farmacológico , Ratones , Microambiente TumoralRESUMEN
Specific mechanisms by which tumor-infiltrating lymphocytes (TIL) become dysfunctional remain poorly understood. Here, we employed a two-pronged approach using single-cell mass cytometry and tissue imaging technologies to dissect TILs from 25 patients with resectable and 35 patients with advanced non-small cell lung cancer (NSCLC). We identified a burned-out CD8+ TIL subset (Ebo) that specifically accumulated within the tumor microenvironment (TME) but not in adjacent nontumoral tissues. Ebo showed the highest expression of proliferation and activation markers but produced the lowest amount of IFNγ and were the most apoptotic CD8+ TIL subset. Using a humanized patient-derived tumor xenograft model, we demonstrated that Ebo expansion occurred within the TME in a PD-1/B7-H1 pathway-dependent manner. Ebo abundance in baseline tumor tissues was associated with resistance to anti-PD therapy in patients with NSCLC. Our study identifies a dysfunctional TIL subset, with distinct features from previously described exhausted T cells, and implies strategies to overcome immunotherapy resistance. SIGNIFICANCE: We identified a highly proliferative, overactivated, and apoptotic dysfunctional CD8+ tumor-infiltrating subpopulation that is functionally distinct from previously described exhausted T cells. This population is expanded in lung cancer tissues in a PD-1/B7-H1-dependent manner, and its abundance is associated with resistance to cancer immunotherapy, thus becoming a potential tissue biomarker.This article is highlighted in the In This Issue feature, p. 1601.
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Linfocitos T CD8-positivos , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/patología , Microambiente Tumoral , Anciano , Animales , Carcinoma de Pulmón de Células no Pequeñas/terapia , Femenino , Humanos , Inmunoterapia , Neoplasias Pulmonares/terapia , Masculino , Ratones , Ratones Endogámicos NOD , Estudios ProspectivosRESUMEN
Systemic lupus erythematosus (SLE) and discoid lupus erythematosus (DLE) of the skin are autoimmune diseases characterized by inappropriate immune responses against self-proteins; the key elements that determine disease pathogenesis and progression are largely unknown. Here, we show that mice lacking immune inhibitory receptor VISTA or programmed death-1 homolog (PD-1H KO) on a BALB/c background spontaneously develop cutaneous and systemic autoimmune diseases resembling human lupus. Cutaneous lupus lesions of PD-1H KO mice have clustering of plasmacytoid dendritic cells (pDCs) similar to human DLE. Using mass cytometry, we identified proinflammatory neutrophils as critical early immune infiltrating cells within cutaneous lupus lesions of PD-1H KO mice. We also found that PD-1H is highly expressed on immune cells in human SLE, DLE lesions, and cutaneous lesions of MRL/lpr mice. A PD-1H agonistic monoclonal antibody in MRL/lpr mice reduces cutaneous disease, autoantibodies, inflammatory cytokines, chemokines, and immune cell expansion. Furthermore, PD-1H on both T cells and myeloid cells including neutrophils and pDCs could transmit inhibitory signals, resulting in reduced activation and function, establishing PD-1H as an inhibitory receptor on T cells and myeloid cells. On the basis of these findings, we propose that PD-1H is a critical element in the pathogenesis and progression of lupus, and PD-1H activation could be effective for treatment of systemic and cutaneous lupus.
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Autoinmunidad , Lupus Eritematoso Cutáneo/inmunología , Lupus Eritematoso Sistémico/inmunología , Proteínas de la Membrana/metabolismo , Animales , Artritis/patología , Autoanticuerpos/inmunología , Células Dendríticas/inmunología , Humanos , Inflamación/patología , Interferón Tipo I/metabolismo , Proteínas de la Membrana/agonistas , Proteínas de la Membrana/deficiencia , Ratones Endogámicos BALB C , Ratones Endogámicos MRL lpr , Células Mieloides/metabolismo , Neutrófilos/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Terpenos , Regulación hacia ArribaRESUMEN
Methylene diphenyl diisocyanate (MDI), the most abundantly produced diisocyanate worldwide, is among the best recognized chemical causes of occupational asthma. The bulk of synthesized MDI, the 4,4' isomer, has been the focus of most biochemical research to date. The biological reactivity of other MDI isomers (2,2' and 2,4'), present at concentrations approaching 50% in some commercial products, remains less clear. We hypothesized 2,2' and 2,4' MDI react with glutathione (GSH), a major anti-oxidant of the lower airways, similarly to 4,4' MDI, and that the products could be characterized using a combination of LC-UV-MS and MS/MS. Purified 2,2' and 2,4' MDI isomers were mixed with GSH in pH-buffered aqueous phase at 37°C and reaction products were analyzed at varying time points. Within minutes, S-linked bis(GSH)-MDI conjugates were detectable as the dominant [M+H]+ ion, with an 865.25 m/z and more intense [M+2H]2+ ions of the same nominal mass. Upon longer reaction, [M+H]+ ions with greater retention times and the 558.17 m/z expected for mono(GSH)-MDI reaction products were observed, and exhibited MS/MS collision-induced dissociation (CID)-fragmentation patterns consistent with cyclized structures. Compared with 4,4' MDI, 2,2' and 2,4' isomers exhibit similar rapid reactivity with GSH and formation of bis(GSH)-MDI conjugates, but greater formation of cyclized mono(GSH) conjugates following extended reaction times (10 minutes to 2 hours). Further translational studies will be required to determine if the present in vitro findings extend to the complex lower airway microenvironment in vivo.
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Biomonitoring of methylene diphenyl diisocyanate (MDI) in urine may be useful in industrial hygiene and exposure surveillance approaches toward disease (occupational asthma) prevention and in understanding pathways by which the internalized chemical is excreted. We explored possible urine biomarkers of MDI exposure in mice after respiratory tract exposure to MDI, as glutathione (GSH) reaction products (MDI-GSH), and after skin exposure to MDI dissolved in acetone. LC-MS analyses of urine identified a unique m/ z 543.29 [M + H]+ ion from MDI-exposed mice but not from controls. The m/ z 543.29 [M + H]+ ion was detectable within 24 h of a single MDI skin exposure and following multiple respiratory tract exposures to MDI-GSH reaction products. The m/ z 543.29 [M + H]+ ion possessed properties of dilysine-MDI, including (a) an isotope distribution pattern for a molecule with the chemical formula C27H38N6O6, (b) the expected collision-induced dissociation (CID) fragmentation pattern upon MS/MS, and (c) a retention time in reversed-phase LC-MS identical to that of synthetic dilysine-MDI. Further MDI-specific Western blot studies suggested albumin (which contains multiple dilysine sites susceptible to MDI carbamylation) as a possible source for dilysine-MDI and the presence of MDI-conjugated albumin in urine up to 6 days after respiratory tract exposure. Two additional [M + H]+ ions ( m/ z 558.17 and 863.23) were found exclusively in urine of mice exposed to MDI-GSH via the respiratory tract and possessed characteristics of previously described cyclized MDI-GSH and oxidized glutathione (GSSG)-MDI conjugates, respectively. Together the data identify urinary biomarkers of MDI exposure in mice and possible guidance for future translational investigation.
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Dipéptidos/orina , Isocianatos/orina , Piel/química , Animales , Biomarcadores/orina , Cromatografía Liquida , Dipéptidos/administración & dosificación , Dipéptidos/química , Glutatión/química , Glutatión/orina , Isocianatos/administración & dosificación , Isocianatos/química , Ratones , Estructura Molecular , Espectrometría de Masas en TándemRESUMEN
The objective of this study was to validate a method for the determination of laromustine (VNP40101M) and short-lived its active metabolite (VNP4090CE) that has a half-life in human blood of <90 s in human plasma by liquid chromatography (LC) with tandem mass spectrometric (MS/MS) detection. We overcome the stability dilemma by acidified the human plasma with citric acid. Laromustine "breaks" down on the source of mass spectrometry to give m/z 249 which is the same m/z for VNP4090CE. Because VNP4090CE and laromustine elute at approximate retention time of 1.93 and 2.94 min, respectively, we were able to quantify both of them in one method. VNP40101M, VNP4090CE and the internal standards were extracted from human plasma by liquid-liquid extraction into ethyl ether. The ethyl ether layer was evaporated, reconstituted and analyzed using LC with MS/MS detection. Validation parameters such as selectivity, limit of quantitation, linearity, precision, accuracy, recovery, autosampler viability, freeze-thaw cycles and compounds stability are evaluated for this method. Results were calculated using peak area ratios, and calibration curves were generated using a weighted (1/x2) linear least-squares regression. Calibration curves for VNP40101M and VNP4090CE in human plasma ranged from 1.00 to 1,000 ng/mL. In this study, both intra- and inter-assay results demonstrated a relative standard deviation for calibration standards (inter-assay) and quality control samples (intra- and inter-assay) to be ≤15.0%. In this method, there is ~1.79% isotopic interference of VNP40101M to VNP40101M-IS, and ~3.76% isotopic interference of VNP4090CE to VNP4090CE-IS. It was concluded that there was no significant carryover.
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Cromatografía Liquida/métodos , Hidrazinas/sangre , Sulfonamidas/sangre , Espectrometría de Masas en Tándem/métodos , Humanos , Hidrazinas/química , Límite de Detección , Modelos Lineales , Reproducibilidad de los Resultados , Sulfonamidas/químicaRESUMEN
1. Hexamethylenediisocyanate (HDI) is a widely used aliphatic diisocyanate and a well-recognized cause of occupational asthma. 2. "Self" molecules (peptides/proteins) in the lower airways, susceptible to chemical reactivity with HDI, have been hypothesized to play a role in asthma pathogenesis and/or chemical metabolism, but remain poorly characterized. 3. This study employed unique approaches to identify and characterize "self" targets of HDI reactivity in the lower airways. Anesthetized rabbits free breathed through a tracheostomy tube connected to chambers containing either, O2, or O2 plus â¼200 ppb HDI vapors. Following 60 minutes of exposure, the airways were lavaged and the fluid was analyzed by LC-MS and LC-MS/MS. 4. The low-molecular weight (<3 kDa) fraction of HDI exposed, but not control rabbit bronchoalveolar lavage (BAL) fluid identified 783.26 and 476.18 m/z [M+H]+ ions with high energy collision-induced dissociation (HCD) fragmentation patterns consistent with bis glutathione (GSH)-HDI and mono(GSH)-HDI. Proteomic analyses of the high molecular weight (>3 kDa) fraction of exposed rabbit BAL fluid identified HDI modification of specific lysines in uteroglobin (aka clara cell protein) and albumin. 5. In summary, this study utilized a unique approach to chemical vapor exposure in rabbits, to identify HDI reaction products with "self" molecules in the lower airways.
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Isocianatos/farmacología , Pulmón/efectos de los fármacos , Traqueostomía , Secuencia de Aminoácidos , Animales , Líquidos Corporales/metabolismo , Cromatografía Liquida , Masculino , Espectrometría de Masas , Modelos Moleculares , Peso Molecular , Péptidos/química , Conejos , VolatilizaciónRESUMEN
Hexamethylene diisocyanate (HDI) is an important industrial chemical that can cause asthma, however pathogenic mechanisms remain unclear. Upon entry into the respiratory tract, HDI's N=C=O groups may undergo nucleophilic addition (conjugate) to host molecules (e.g. proteins), or instead react with water (hydrolyze), releasing CO2 and leaving a primary amine in place of the original N=C=O. We hypothesized that (primary amine groups present on) hydrolyzed or partially hydrolyzed HDI may compete with proteins and water as a reaction target for HDI in solution, resulting in polymers that could be identified and characterized using LC-MS and LC-MS/MS. Analysis of the reaction products formed when HDI was mixed with a pH buffered, isotonic, protein containing solution identified multiple [M+H]+ ions with m/z's and collision-induced dissociation (CID) fragmentation patterns consistent with those expected for dimers (259.25/285.23 m/z), and trimers (401.36/427.35 m/z) of partially hydrolyzed HDI (e.g. ureas/oligoureas). Human peripheral blood mononuclear cells (PBMCs) and monocyte-like U937, but not airway epithelial NCI-H292 cell lines cultured with these HDI ureas contained a novel 260.23 m/z [M+H]+ ion. LC-MS/MS analysis of the 260.23 m/z [M+H]+ ion suggest the formula C13H29N3O2 and a structure containing partially hydrolyzed HDI, however definitive characterization will require further orthogonal analyses.
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Isocianatos/química , Células Cultivadas , Cromatografía Liquida , Humanos , Iones/síntesis química , Iones/química , Isocianatos/síntesis química , Espectrometría de Masas , Estructura Molecular , Polimerizacion , SolucionesRESUMEN
Analysis of multiplexed assays is highly important for clinical diagnostics and other analytical applications. Mass cytometry enables multi-dimensional, single-cell analysis of cell type and state. In mass cytometry, the rare earth metals used as reporters on antibodies allow determination of marker expression in individual cells. Barcode-based bioassays for CyTOF are able to encode and decode for different experimental conditions or samples within the same experiment, facilitating progress in producing straightforward and consistent results. Herein, an integrated protocol for automated sample preparation for barcoding used in conjunction with mass cytometry for clinical bioanalysis samples is described; we offer results of our work with barcoding protocol optimization. In addition, we present some points to be considered in order to minimize the variability of quantitative mass cytometry measurements. For example, we discuss the importance of having multiple populations during titration of the antibodies and effect of storage and shipping of labelled samples on the stability of staining for purposes of CyTOF analysis. Data quality is not affected when labelled samples are stored either frozen or at 4 °C and used within 10 days; we observed that cell loss is greater if cells are washed with deionized water prior to shipment or are shipped in lower concentration. Once the labelled samples for CyTOF are suspended in deionized water, the analysis should be performed expeditiously, preferably within the first hour. Damage can be minimized if the cells are resuspended in phosphate-buffered saline (PBS) rather than deionized water while waiting for data acquisition.
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Automatización , Citometría de Flujo/métodos , Análisis de la Célula Individual/métodos , Manejo de Especímenes , Anticuerpos/análisis , HumanosRESUMEN
1. Alkylating agents are capable of introducing an alkyl group into nucleophilic sites on DNA or RNA through covalent bond. Laromustine is an active member of a relatively new class of sulfonylhydrazine prodrugs under development as antineoplastic alkylating agents, and displays significant single-agent activity. 2. This is the first report of the population pharmacokinetic analysis of laromustine, 106 patients, 66 with hematologic malignancies and 40 with solid tumors, participated in five clinical trials worldwide. Of these, 104 patients were included in the final NONMEM analysis. 3. The population estimates for total clearance (CL) and volume of distribution of the central compartment (V1) were 96.3 L/h and 45.9 L, associated with high inter-patient variability of 52.9% and 79.8% and inter-occasion variability of 26.7% and 49.3%, respectively. The population estimates for Q and V2 were 73.2 L/h and 29.9 L, and inter-patient variability in V2 was 63.1%, respectively. 4. The estimate of Vss (75.8 L) exceeds total body water, indicating that laromustine is distributed to tissues. The half-life is short, less than 1 h, reflecting rapid clearance. Population PK analysis showed laromustine pharmacokinetics to be independent of dose and organ function with no effect on subsequent dosing cycles.
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Antineoplásicos Alquilantes/farmacocinética , Hidrazinas/farmacocinética , Sulfonamidas/farmacocinética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Modelos Biológicos , ProfármacosRESUMEN
Metabolomics is a relatively new and rapidly growing area of post-genomic biological research. As use of metabolomics technology grows throughout the spectrum of drug discovery and development, and its applications broaden, its impact is expanding dramatically. This review seeks to provide the reader with a brief history of the development of metabolomics, its significance and strategies for conducting metabolomics studies. The most widely used analytical tools for metabolomics: NMR, LC-MS and GC-MS, are discussed along with considerations for their use. Herein, we will show how metabolomics can assist in pharmaceutical research studies, such as pharmacology and toxicology, and discuss some examples of the importance of metabolomics analysis in research and development.
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Metabolómica , Investigación Farmacéutica , Animales , Cromatografía Liquida , Descubrimiento de Drogas , Humanos , Espectrometría de MasasAsunto(s)
Citofotometría/métodos , Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Espectrometría de Masas/métodos , Animales , Citofotometría/instrumentación , Descubrimiento de Drogas/instrumentación , Ensayos Analíticos de Alto Rendimiento/instrumentación , Humanos , Espectrometría de Masas/instrumentación , Paladio/químicaRESUMEN
PURPOSE: It has become quite clear that single cancer biomarkers cannot in general provide high sensitivity and specificity for reliable clinical cancer diagnostics. This paper explores the feasibility of rapid detection of multiple biomarker proteins in model oral cancer samples using label-free protein relative quantitation. EXPERIMENTAL DESIGN: MS-based label-free quantitative proteomics offer a rapid alternative that bypasses the need for stable isotope containing compounds to chemically bind and label proteins. Total protein content in oral cancer cell culture conditioned media was precipitated, subjected to proteolytic digestion, and then analyzed using a nano-UPLC (where UPLC is ultra-performance liquid chromatography) coupled to a hybrid Q-Tof ion-mobility mass spectrometry (MS). RESULTS: Rapid, simultaneous identification and quantification of multiple possible cancer biomarker proteins was achieved. In a comparative study between cancer and noncancer samples, approximately 952 proteins were identified using a high-throughput 1D ion mobility assisted data independent acquisition (IM-DIA) approach. As we previously demonstrated that interleukin-8 (IL-8) and vascular endothelial growth factor A (VEGF-A) were readily detected in oral cancer cell conditioned media(1), we targeted these biomarker proteins to validate our approach. Target biomarker protein IL-8 was found between 3.5 and 8.8 fmol, while VEGF-A was found at 1.45 fmol in the cancer cell media. CONCLUSIONS AND CLINICAL RELEVANCE: Overall, our data suggest that the nano-UPLC-IM-DIA bioassay is a feasible approach to identify and quantify proteins in complex samples without the need for stable isotope labeling. These results have significant implications for rapid tumor diagnostics and prognostics by monitoring proteins such as IL-8 and VEGF-A implicated in cancer development and progression. The analysis in tissue or plasma is not possible at this time, but the subsequent work would be needed for validation.
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Biomarcadores de Tumor/análisis , Espectrometría de Masas/métodos , Neoplasias de la Boca/diagnóstico , Neoplasias de la Boca/metabolismo , Proteínas de Neoplasias/análisis , Cromatografía Líquida de Alta Presión , Humanos , Nanotecnología , Células Tumorales CultivadasRESUMEN
Isocyanates differ from many other xenobiotics in their ability to form S-linked conjugates with glutathione (GSH) through direct nucleophilic addition reactions (e.g. without enzymatic "preactivation" and/or transferase activity), potentially predisposing them to metabolism via the mercapturic acid pathway. In vivo, mono-isocyanates are metabolized via the mercapturic acid pathway and excreted as N-acetylated cysteine conjugates, however, the metabolism of di-isocyanates remains unclear. We assessed the ability of purified human gamma-glutamyl transpeptidase-1 (GGT-1), a primary enzyme of the mercapturic acid pathway, to cleave S-linked GSH conjugates of 4,4'-methylene diphenyl diisocyanate (MDI) and 1,6-hexamethylene diisocyanate (HDI), two widely used industrial chemicals. A combination of liquid chromatography (LC), tandem mass spectrometry (MS/MS) and hydrogen-deuterium exchange studies confirmed GGT-1 mediated formation of the 607.2 and 525.2 m/z (M + H)(+) ions corresponding to bis(cys-gly)-MDI and bis(cys-gly)-HDI, respectively, the cleavage products expected from the corresponding bis(GSH)-diisocyanate conjugates. Additional intermediate metabolites and mono(cys-gly)-conjugates with partially hydrolyzed diisocyanate were also observed. Consistent with GGT enzyme kinetics, metabolism proceeded more rapidly under conditions that favored transpeptidation versus hydrolytic mechanisms of cleavage. Together the data demonstrate the capacity of human GGT-1 to cleave GSH conjugates of both aromatic and aliphatic diisocyanates, suggesting a potential role in their metabolism.
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Isocianatos/toxicidad , Xenobióticos/toxicidad , gamma-Glutamiltransferasa/metabolismo , Acetilcisteína/metabolismo , Glutatión/metabolismo , HumanosRESUMEN
Cytometry by time-of-flight (CyTOF) is a novel technology for the real-time analysis of single cells. CyTOF is a significant advance in fields including immunology, hematology, and oncology. It resolves multiple metal-conjugated probes per cell with minimal signal overlap, which maximizes the information obtained from each individual sample. CyTOF provides the ability to phenotypically and functionally profile cells from normal and diseased states. Single cell technologies enable researchers to measure the effects of a drug at the single cell level and better understand its mechanism of action. Here, we discuss novel instruments for the analysis of individual biological cells, the impact of recent innovations in support of drug development, and the important roles of CyTOF in drug profiling.
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Diseño de Fármacos , Descubrimiento de Drogas/métodos , Citometría de Flujo/métodos , Animales , Humanos , Análisis de la Célula Individual/métodosRESUMEN
1. Laromustine (VNP40101M, also known as Cloretazine) is a novel sulfonylhydrazine alkylating (anticancer) agent. This article describes the use of quantitative whole-body autoradiography (QWBA) and mass balance to study the tissue distribution, the excretion mass balance and pharmacokinetics after intravenous administration of [(14)C]VNP40101M to rats. A single 10 mg/kg IV bolus dose of [(14)C]VNP40101M was given to rats. 2. The recovery of radioactivity from the Group 1 animals over a 7-day period was an average of 92.1% of the administered dose, which was accounted for in the excreta and carcass. Most of the radioactivity was eliminated within 48 h via urine (48%), with less excreted in feces (5%) and expired air accounted for (11%). The plasma half-life of [(14)C]laromustine was approximately 62 min and the peak plasma concentration (Cmax) averaged 8.3 µg/mL. 3. The QWBA study indicated that the drug-derived radioactivity was widely distributed to tissues through 7 days post-dose after a single 10 mg/kg IV bolus dose of [(14)C]VNP40101M to male pigmented Long-Evans rats. The maximum concentrations were observed at 0.5 or 1 h post-dose for majority tissues (28 of 42). The highest concentrations of radioactivity were found in the small intestine contents at 0.5 h (112.137 µg equiv/g), urinary bladder contents at 3 h (89.636 µg equiv/g) and probably reflect excretion of drug and metabolites. The highest concentrations in specific organs were found in the renal cortex at 1 h (28.582 µg equiv/g), small intestine at 3 h (16.946 µg equiv/g), Harderian gland at 3 h (12.332 µg equiv/g) and pancreas at 3 h (12.635 µg equiv/g). Concentrations in the cerebrum (1.978 µg equiv/g), cerebellum (2.109 µg equiv/g), medulla (1.797 µg equiv/g) and spinal cord (1.510 µg equiv/g) were maximal at 0.5 h post-dose and persisted for 7 days. 4. The predicted total body and target organ exposures for humans given a single 100 µCi IV dose of [(14)C]VNP40101M were well within the medical guidelines for maximum radioactivity exposures in human subjects.