RESUMEN
The selection and prioritization of pharmaceuticals and their transformation products for evaluating effects on the environment and human health is a challenging task. One common approach is based on compounds (e.g., mixture composition, concentrations), and another on biology (e.g., relevant endpoint, biological organizational level). Both of these approaches often resemble a Lernaean Hydra-they can create more questions than answers. The present study embraces this complexity, providing an integrated approach toward assessing the potential effects of transformation products of pharmaceuticals by means of mutagenicity, estrogenicity, and differences in the gene expression profiles. Mutagenicity using the tk kinase assay was applied to assess a list of 11 priority pharmaceuticals, namely, atenolol, azithromycin, carbamazepine, diclofenac, ibuprofen, erythromycin, metoprolol, ofloxacin, propranolol, sulfamethoxazole, and trimethoprim. The most mutagenic compounds were found to be ß-blockers. In parallel, the photolabile pharmaceuticals were assessed for their mixture effects on mutagenicity (tk assay), estrogenicity (T47D- KBluc assay), and gene expression (microarrays). Interestingly, the mixtures were mutagenic at the µg/L level, indicating a synergistic effect. None of the photolysed mixtures were statistically significantly estrogenic. Gene expression profiling revealed effects related mainly to certain pathways, those of the p53 gene, mitogen-activated protein kinase, alanine, aspartate, and glutamate metabolism, and translation-related (spliceosome). Fourteen phototransformation products are proposed based on the m/z values found through ultra-performance liquid chromatography-tandem mass spectrometry analysis. The transformation routes of the photolysed mixtures indicate a strong similarity with those obtained for each pharmaceutical separately. This finding reinforces the view that transformation products are to be expected in naturally occurring mixtures. Environ Toxicol Chem 2016;35:2753-2764. © 2016 SETAC.
Asunto(s)
Antagonistas Adrenérgicos beta/toxicidad , Mezclas Complejas/toxicidad , Mutágenos/toxicidad , Preparaciones Farmacéuticas/análisis , Transcriptoma/efectos de los fármacos , Contaminantes Químicos del Agua/toxicidad , Antagonistas Adrenérgicos beta/análisis , Antagonistas Adrenérgicos beta/efectos de la radiación , Animales , Línea Celular Tumoral , Mezclas Complejas/análisis , Mezclas Complejas/efectos de la radiación , Perfilación de la Expresión Génica , Humanos , Ratones , Análisis por Micromatrices , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutágenos/análisis , Mutágenos/efectos de la radiación , Ofloxacino , Preparaciones Farmacéuticas/efectos de la radiación , Fotólisis , Contaminantes Químicos del Agua/análisis , Contaminantes Químicos del Agua/efectos de la radiaciónRESUMEN
Smoking is the main risk factor for urothelial bladder cancer. In former smokers the risk decreases but does not reach the low level of never smokers. This indicates reversible and permanent smoking-derived genetic alterations. Transcriptional changes may point to mechanisms, how smoking promotes urothelial bladder cancer. To identify smoking-derived transcriptional changes we performed gene expression profiling in current, former, and never smokers, using tumor and tumor-free urothelium from patients with nonmuscle-invasive urothelial bladder cancer (NMIBC) or muscle-invasive urothelial bladder cancer (MIBC). Smoking turned out to influence gene expression much less than tumor stage (NMIBC or MIBC) and tumor transformation (tumor-free or tumor). Smoking seemed to influence gene expression in patients with MIBC more strongly compared to those with NMIBC. The least irreversible changes after smoking cessation were proposed in tumor-free urothelium from patients with NMIBC. Growth factors and oncogenes were up-regulated in tumor-free urothelium from smokers with MIBC but not from smokers with NMIBC. A panel of genes up-regulated in smokers have potential for early detection and distinction of MIBC from NMIBC using tumor-free tissue.
Asunto(s)
Regulación de la Expresión Génica , Fumar/efectos adversos , Fumar/genética , Neoplasias de la Vejiga Urinaria/patología , Vejiga Urinaria/patología , Urotelio/patología , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Progresión de la Enfermedad , Humanos , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Transducción de Señal , Fumar/metabolismo , Fumar/patología , Cese del Hábito de Fumar , Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/metabolismo , Urotelio/metabolismoRESUMEN
BACKGROUND: A defective innate immune response may contribute to the pathogenesis of Crohn's disease (CD) and ulcerative colitis (UC). Employing a global gene expression analysis, this study was aimed at identifying specifically regulated genes within the epithelial compartment in inflammatory bowel disease (IBD). METHODS: The epithelial fraction of human ileal mucosa samples from surgical specimens was obtained by laser microdissection. Gene expression was examined by global expression profiling (n = 18, Affymetrix), quantitative reverse-transcription polymerase chain reaction (RT-PCR) (n = 35), immunoblot analysis (n = 9), and immunohistochemistry (n = 25). RESULTS: Global expression profiling revealed a pronounced downregulation of the retinoic acid-inducible gene I (RIG-I) within the epithelial layer of the ileum in patients with CD but not with UC. The downregulation of RIG-I was confirmed by quantitative RT-PCR, immunoblot analysis, and immunohistochemistry. CONCLUSIONS: Epithelial downregulation of RIG-I, a known pattern recognition receptor for viral components, might contribute to alterations of the innate mucosal immune response, particularly in CD.
Asunto(s)
Biomarcadores/análisis , Colitis Ulcerosa/genética , Enfermedad de Crohn/genética , ARN Helicasas DEAD-box/genética , Mucosa Intestinal/metabolismo , Adulto , Anciano , Biomarcadores/metabolismo , Western Blotting , Estudios de Casos y Controles , Estudios de Cohortes , Colitis Ulcerosa/metabolismo , Enfermedad de Crohn/metabolismo , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/metabolismo , Femenino , Perfilación de la Expresión Génica , Humanos , Técnicas para Inmunoenzimas , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , ARN Mensajero/genética , Receptores Inmunológicos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Primary mediastinal B-cell lymphoma (PMBL) and classical Hodgkin lymphoma (cHL) share a frequent constitutive activation of JAK (Janus kinase)/STAT signaling pathway. Because of complex, nonlinear relations within the pathway, key dynamic properties remained to be identified to predict possible strategies for intervention. We report the development of dynamic pathway models based on quantitative data collected on signaling components of JAK/STAT pathway in two lymphoma-derived cell lines, MedB-1 and L1236, representative of PMBL and cHL, respectively. We show that the amounts of STAT5 and STAT6 are higher whereas those of SHP1 are lower in the two lymphoma cell lines than in normal B cells. Distinctively, L1236 cells harbor more JAK2 and less SHP1 molecules per cell than MedB-1 or control cells. In both lymphoma cell lines, we observe interleukin-13 (IL13)-induced activation of IL4 receptor α, JAK2, and STAT5, but not of STAT6. Genome-wide, 11 early and 16 sustained genes are upregulated by IL13 in both lymphoma cell lines. Specifically, the known STAT-inducible negative regulators CISH and SOCS3 are upregulated within 2 hours in MedB-1 but not in L1236 cells. On the basis of this detailed quantitative information, we established two mathematical models, MedB-1 and L1236 model, able to describe the respective experimental data. Most of the model parameters are identifiable and therefore the models are predictive. Sensitivity analysis of the model identifies six possible therapeutic targets able to reduce gene expression levels in L1236 cells and three in MedB-1. We experimentally confirm reduction in target gene expression in response to inhibition of STAT5 phosphorylation, thereby validating one of the predicted targets.
Asunto(s)
Enfermedad de Hodgkin/metabolismo , Interleucina-13/farmacología , Linfoma de Células B/metabolismo , Neoplasias del Mediastino/metabolismo , Modelos Biológicos , Terapia Molecular Dirigida/métodos , Línea Celular Tumoral , Enfermedad de Hodgkin/genética , Enfermedad de Hodgkin/terapia , Humanos , Janus Quinasa 2/antagonistas & inhibidores , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Linfoma de Células B/genética , Linfoma de Células B/terapia , Neoplasias del Mediastino/genética , Neoplasias del Mediastino/terapia , Fosforilación/efectos de los fármacos , Factor de Transcripción STAT5/metabolismo , Factor de Transcripción STAT6/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/metabolismoRESUMEN
BACKGROUND: External stimulations of cells by hormones, cytokines or growth factors activate signal transduction pathways that subsequently induce a re-arrangement of cellular gene expression. The analysis of such changes is complicated, as they consist of multi-layered temporal responses. While classical analyses based on clustering or gene set enrichment only partly reveal this information, matrix factorization techniques are well suited for a detailed temporal analysis. In signal processing, factorization techniques incorporating data properties like spatial and temporal correlation structure have shown to be robust and computationally efficient. However, such correlation-based methods have so far not be applied in bioinformatics, because large scale biological data rarely imply a natural order that allows the definition of a delayed correlation function. RESULTS: We therefore develop the concept of graph-decorrelation. We encode prior knowledge like transcriptional regulation, protein interactions or metabolic pathways in a weighted directed graph. By linking features along this underlying graph, we introduce a partial ordering of the features (e.g. genes) and are thus able to define a graph-delayed correlation function. Using this framework as constraint to the matrix factorization task allows us to set up the fast and robust graph-decorrelation algorithm (GraDe). To analyze alterations in the gene response in IL-6 stimulated primary mouse hepatocytes, we performed a time-course microarray experiment and applied GraDe. In contrast to standard techniques, the extracted time-resolved gene expression profiles showed that IL-6 activates genes involved in cell cycle progression and cell division. Genes linked to metabolic and apoptotic processes are down-regulated indicating that IL-6 mediated priming renders hepatocytes more responsive towards cell proliferation and reduces expenditures for the energy metabolism. CONCLUSIONS: GraDe provides a novel framework for the decomposition of large-scale 'omics' data. We were able to show that including prior knowledge into the separation task leads to a much more structured and detailed separation of the time-dependent responses upon IL-6 stimulation compared to standard methods. A Matlab implementation of the GraDe algorithm is freely available at http://cmb.helmholtz-muenchen.de/grade.