RESUMEN
BACKGROUND: Many cancer patients do not obtain clinical benefit from immune checkpoint inhibition. Checkpoint blockade targets T cells, suggesting that tyrosine kinase activity profiling of baseline peripheral blood mononuclear cells may predict clinical outcome. METHODS: Here a total of 160 patients with advanced melanoma or non-small-cell lung cancer (NSCLC), treated with anti-cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA-4) or anti-programmed cell death 1 (anti-PD-1), were divided into five discovery and cross-validation cohorts. The kinase activity profile was generated by analyzing phosphorylation of peripheral blood mononuclear cell lysates in a microarray comprising of 144 peptides derived from sites that are substrates for protein tyrosine kinases. Binary grouping into patients with or without clinical benefit was based on Response Evaluation Criteria in Solid Tumors V.1.1. Predictive models were trained using partial least square discriminant analysis (PLS-DA), performance of the models was evaluated by estimating the correct classification rate (CCR) using cross-validation. RESULTS: The kinase phosphorylation signatures segregated responders from non-responders by differences in canonical pathways governing T-cell migration, infiltration and co-stimulation. PLS-DA resulted in a CCR of 100% and 93% in the anti-CTLA-4 and anti-PD1 melanoma discovery cohorts, respectively. Cross-validation cohorts to estimate the accuracy of the predictive models showed CCRs of 83% for anti-CTLA-4 and 78% or 68% for anti-PD-1 in melanoma or NSCLC, respectively. CONCLUSION: Blood-based kinase activity profiling for response prediction to immune checkpoint inhibitors in melanoma and NSCLC revealed increased kinase activity in pathways associated with T-cell function and led to a classification model with a highly accurate classification rate in cross-validation groups. The predictive value of kinase activity profiling is prospectively verified in an ongoing trial.
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Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inmunoterapia/métodos , Neoplasias/tratamiento farmacológico , Adulto , Anciano , Femenino , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Neoplasias/patologíaRESUMEN
Estrogen receptor alpha (ERα) and retinoic acid receptors (RARs) play important and opposite roles in breast cancer growth. While exposure to ERα agonists such as 17ß-estradiol (E2) is related to proliferation, RAR agonists such as all-trans retinoic acid (AtRA) induce anti-proliferative effects. Although crosstalk between these pathways has been proposed, the molecular mechanisms underlying this interplay are still not completely unraveled. The aim of this study was to evaluate the effects of AtRA on ERα-mediated signaling in the ERα positive cell lines MCF7/BUS and U2OS-ERα-Luc to investigate some of the possible underlying modes of action. To do so, this study assessed the effects of AtRA on different ERα-related events such as ERα-mediated cell proliferation and gene expression, ERα-coregulator binding and ERα subcellular localization. AtRA-mediated antagonism of E2-induced signaling was observed in the proliferation and gene expression studies. However, AtRA showed no remarkable effects on the E2-driven coregulator binding and subcellular distribution of ERα. Interestingly, in the absence of E2, ERα-mediated gene expression, ERα-coregulator binding and ERα subcellular mobilization were increased upon exposure to micromolar concentrations of AtRA found to inhibit cell proliferation after long-term exposure. Nevertheless, experiments using purified ERα showed that direct binding of AtRA to ERα does not occur. Altogether, our results using MCF7/BUS and U2OS-ERα-Luc cells suggest that AtRA, without being a direct ligand of ERα, can indirectly interfere on basal ERα-coregulator binding and basal ERα subcellular localization in addition to the previously described crosstalk mechanisms such as competition of ERs and RARs for DNA binding sites.
Asunto(s)
Estrógenos/farmacología , Receptores de Estrógenos/metabolismo , Transducción de Señal , Tretinoina/farmacología , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Humanos , Luciferasas/metabolismo , Células MCF-7 , Unión Proteica/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Retinoic Acid Receptor alpha (RARα/NR1B1), Retinoic Acid Receptor beta (RARß/NR1B2) and Retinoic Acid Receptor gamma (RARγ/NR1B3) are transcription factors regulating gene expression in response to retinoids. Within the RAR genomic pathways, binding of RARs to coregulators is a key intermediate regulatory phase. However, ligand-dependent interactions between the wide variety of coregulators that may be present in a cell and the different RAR subtypes are largely unknown. The aim of this study is to characterize the coregulator binding profiles of RARs in the presence of the pan-agonist all-trans-Retinoic Acid (AtRA); the subtype-selective agonists Am80 (RARα), CD2314 (RARß) and BMS961 (RARγ); and the antagonist Ro415253. To this end, we used a microarray assay for coregulator-nuclear receptor interactions to assess RAR binding to 154 motifs belonging to >60 coregulators. The results revealed a high number of ligand-dependent RAR-coregulator interactions among all RAR variants, including many binding events not yet described in literature. Next, this work confirmed a greater ligand-independent activity of RARß compared to the other RAR subtypes based on both higher basal and lower ligand-driven coregulator binding. Further, several coregulator motifs showed selective binding to a specific RAR subtype. Next, this work showed that subtype-selective agonists can be successfully discriminated by using coregulator binding assays. Finally this study demonstrated the possible applications of a coregulator binding assay as a tool to discriminate between agonistic/antagonistic actions of ligands. The RAR-coregulator interactions found will be of use to direct further studies to better understand the mechanisms driving the eventual actions of retinoids.
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Receptores de Ácido Retinoico/química , Receptor alfa de Ácido Retinoico/química , Secuencias de Aminoácidos , Antracenos/farmacología , Benzoatos/farmacología , Sitios de Unión , Cromanos , Análisis por Matrices de Proteínas , Unión Proteica , Dominios Proteicos , Receptores de Ácido Retinoico/agonistas , Receptores de Ácido Retinoico/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Elementos de Respuesta , Receptor alfa de Ácido Retinoico/agonistas , Receptor alfa de Ácido Retinoico/antagonistas & inhibidores , Retinoides/farmacología , Relación Estructura-Actividad , Tetrahidronaftalenos/farmacología , Tiofenos/farmacología , Tretinoina/farmacología , Receptor de Ácido Retinoico gammaRESUMEN
The aim of the present study was to investigate modulation of the interaction of ERα and ERß with coregulators in the ligand dependent responses induced by the ER antagonistic compounds 4OHT and fulvestrant. Comparison with the modulation index (MI) profiles for the ER agonist estradiol (E2) will elucidate whether differences in the (ant)agonist dependent interaction of ERα and ERß with coregulators expressed in MI profiles contribute to the differences in (ant)agonist responses. To this end, the selected ER antagonistic compounds were first characterized for intrinsic relative potency and efficacy towards ERα and ERß using ER selective U2OS reporter gene assays, and subsequently tested for ligand dependent modulation of the interaction of ERα and ERß with coregulators using the MARCoNI assay. Results obtained indicate a preference of 4OHT to antagonize ERß and find fulvestrant to be less ER specific. MARCoNI assay responses reveal that ERα and ERß mediated interaction with coregulators expressed in MI profiles are similar for 4OHT and fulvestrant and generally opposite to the MI profile of the ER agonist E2. Hierarchical clustering based on the MI profiles appeared able to clearly discriminate the two compounds with ER antagonistic properties from the ER agonist E2. Taken together the data reveal that modulation of the interaction of ERs with coregulators discriminates ER agonists from antagonists but does not discriminate between the less specific ER antagonist fulvestrant and the preferential ERß antagonistic compound 4OHT. It is concluded that differences in modulation of the interaction of ERα and ERß with coregulators contribute to the differences in ligand dependent responses induced by ER agonists and ER antagonists but the importance of the subtle differences in modulation of the interaction of ERs with coregulators between the ER antagonistic compounds 4OHT and fulvestrant for the ultimate biological effect remains to be established.
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Estradiol/análogos & derivados , Tamoxifeno/análogos & derivados , Línea Celular , Estradiol/farmacología , Moduladores de los Receptores de Estrógeno/farmacología , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/genética , Receptor beta de Estrógeno/metabolismo , Fulvestrant , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Análisis por Micromatrices , Unión Proteica/efectos de los fármacos , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Tamoxifeno/farmacologíaRESUMEN
The aim of the present study was to investigate modulation of the interaction of the ERα and ERß with coregulators in the ligand responses induced by estrogenic compounds. To this end, selective ERα and ERß agonists were characterized for intrinsic relative potency reflected by EC50 and maximal efficacy towards ERα and ERß mediated response in ER selective reporter gene assays, and subsequently tested for induction of cell proliferation in T47D-ERß cells with variable ERα/ERß ratio, and finally for ligand dependent modulation of the interaction of ERα and ERß with coregulators using the MARCoNI assay, with 154 unique nuclear receptor coregulator peptides derived from 66 different coregulators. Results obtained reveal an important influence of the ERα/ERß ratio and receptor selectivity of the compounds tested on induction of cell proliferation. ERα agonists activate cell proliferation whereas ERß suppresses ERα mediated cell proliferation. The responses in the MARCoNI assay reveal that upon ERα or ERß activation by a specific agonist, the modulation of the interaction of the ERs with coregulators is very similar indicating only a limited number of differences upon ERα or ERß activation by a specific ligand. Differences in the modulation of the interaction of the ERs with coregulators between the different agonists were more pronounced. Based on ligand dependent differences in the modulation of the interaction of the ERs with coregulators, the MARCoNI assay was shown to be able to classify the ER agonists discriminating between different agonists for the same receptor, a characteristic not defined by the ER selective reporter gene or proliferation assays. It is concluded that the ultimate effect of the model compounds on proliferation of estrogen responsive cells depends on the intrinsic relative potency of the agonist towards ERα and ERß and the cellular ERα/ERß ratio whereas differences in the modulation of the interaction of the ERα and ERß with coregulators contribute to the ligand dependent responses induced by estrogenic compounds.
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Neoplasias de la Mama/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Receptor alfa de Estrógeno/agonistas , Receptor beta de Estrógeno/agonistas , Estrógenos/farmacología , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Western Blotting , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/genética , Receptor beta de Estrógeno/metabolismo , Femenino , Humanos , Células Tumorales CultivadasRESUMEN
This study investigates whether the previous observation that quercetin increases the transport of PhIP through Caco-2 monolayers in vitro could be confirmed in an in vivo rat model. Co-administration of 1.45 micromol PhIP/kg bw and 30 micromol quercetin/kg bw significantly increased the blood AUC(0-8h) of PhIP in rats to 131+/-14% of the AUC(0-8h) for rats dosed with PhIP alone. Significantly increased blood PhIP levels were detected at 15, 30, 45 and 180 min. At 4 and 8h post-dosing a difference in the PhIP levels in the blood between the two treatment groups was no longer observed. In vitro and in silico modeling of PhIP transport using Caco-2 cells and a previously described kinetic model for PhIP transport revealed that the relative increase in PhIP transport caused by quercetin is dependent on the concentration of the two compounds. When substituting the PhIP and quercetin concentrations used in the in vivo experiment in the kinetic model, an effect of quercetin on PhIP transport was predicted that matches the actual effect of 131% observed in vivo. It is concluded that quercetin increases the bioavailability of the pro-carcinogen PhIP in rats pointing at a potential adverse effect of this supposed beneficial food ingredient.
Asunto(s)
Antioxidantes/farmacología , Carcinógenos/farmacocinética , Imidazoles/farmacocinética , Quercetina/farmacología , Animales , Área Bajo la Curva , Disponibilidad Biológica , Transporte Biológico Activo/efectos de los fármacos , Células CACO-2/metabolismo , Humanos , Masculino , Modelos Biológicos , Distribución Aleatoria , Ratas , Ratas WistarRESUMEN
This study describes and kinetically models the effect of flavonoid mixtures on PhIP transport through Caco-2 monolayers. Previously it was shown that quercetin, luteolin, naringenin and myricetin increase the apical to basolateral PhIP transport in Caco-2 monolayers. In this study, apigenin was shown to exert a similar effect with an apparent K(i) value of 10.8 microM. Additional experiments revealed that several binary flavonoid mixtures and one mixture containing all five model flavonoids increased the apical to basolateral PhIP transport through the Caco-2 monolayer. Assuming competitive inhibition of the apparent active transporter by the flavonoids and concentration-additivity for their inhibiting effect, the kinetic model previously developed to describe the effect of the individual flavonoids on PhIP transport, could be extended and adequately describes the experimental values obtained for the flavonoid mixtures. We conclude that combinations of flavonoids increase the transport of PhIP and do so by interacting in an additive way with the active transport of PhIP. This flavonoid-mediated increase in PhIP transport through Caco-2 monolayers may point at a possible increased bioavailability of PhIP in the presence of flavonoid mixtures in the in vivo situation. This would imply an adverse effect of these supposed beneficial food ingredients.
Asunto(s)
Carcinógenos/farmacocinética , Flavonoides/farmacología , Imidazoles/farmacocinética , Modelos Biológicos , Transporte Biológico Activo/efectos de los fármacos , Células CACO-2 , Sinergismo Farmacológico , HumanosRESUMEN
The present research aimed to study the interaction of three chemicals, methyl mercury, benzene and trichloroethylene, on mRNA expression alterations in rat liver and kidney measured by microarray analysis. These compounds were selected based on presumed different modes of action. The chemicals were administered daily for 14 days at the Lowest-Observed-Adverse-Effect-Level (LOAEL) or at a two- or threefold lower concentration individually or in binary or ternary mixtures. The compounds had strong antagonistic effects on each other's gene expression changes, which included several genes encoding Phase I and II metabolizing enzymes. On the other hand, the mixtures affected the expression of "novel" genes that were not or little affected by the individual compounds. The three compounds exhibited a synergistic interaction on gene expression changes at the LOAEL in the liver and both at the sub-LOAEL and LOAEL in the kidney. Many of the genes induced by mixtures but not by single compounds, such as Id2, Nr2f6, Tnfrsf1a, Ccng1, Mdm2 and Nfkb1 in the liver, are known to affect cellular proliferation, apoptosis and tissue-specific function. This indicates a shift from compound specific response on exposure to individual compounds to a more generic stress response to mixtures. Most of the effects on cell viability as concluded from transcriptomics were not detected by classical toxicological endpoints illustrating the benefit of increased sensitivity of assessing gene expression profiling. These results emphasize the benefit of applying toxicogenomics in mixture interaction studies, which yields biomarkers for joint toxicity and eventually can result in an interaction model for most known toxicants.
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Benceno/toxicidad , Contaminantes Ambientales/toxicidad , Regulación de la Expresión Génica/efectos de los fármacos , Compuestos de Metilmercurio/toxicidad , Tricloroetileno/toxicidad , Animales , Benceno/farmacología , Supervivencia Celular/efectos de los fármacos , Interacciones Farmacológicas , Sinergismo Farmacológico , Contaminantes Ambientales/farmacología , Perfilación de la Expresión Génica/métodos , Riñón/efectos de los fármacos , Riñón/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Compuestos de Metilmercurio/farmacología , Nivel sin Efectos Adversos Observados , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas F344 , Pruebas de Toxicidad , Tricloroetileno/farmacologíaRESUMEN
The nutritional quality of new functional or fortified food products depends on the bioavailability of the nutrient(s) in the human body. Bioavailability is often determined in human intervention studies by measurements of plasma or serum profiles over a certain time period. These studies are time and cost consuming and often appear to lack an optimal study design, leading to follow-up intervention trials. Therefore, an alternative approach is needed that will optimize the development of new products. This study describes an approach to predict human serum concentrations after the consumption of (fortified) food products. The concept is based on the integration of in vitro results with kinetic modeling. As a case study, human serum folate concentrations were predicted after the consumption of folate-fortified milk products for 4 wk. Oral bioavailability was investigated using a step-wise approach in which luminal bioaccessibility and intestinal absorption were independently evaluated. Subsequently, these in vitro data were integrated in a kinetic mathematical (in silico) model to predict serum folate concentrations after the intake of a single dose and during long-term consumption. This approach was evaluated in comparison to a human intervention study in which folic acid-fortified milk products were tested for their effect on serum folate concentrations. A high predictive quality of this alternative in vitro/in silico approach was demonstrated. Finally, this methodology was applied to predict serum folate concentrations after intake of different fortified milk products for 4 wk, showing its benefits for the development of new nutritional products.
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Ácido Fólico/sangre , Absorción Intestinal , Productos Lácteos/análisis , Humanos , Íleon/fisiología , Yeyuno/fisiología , Cinética , Modelos Biológicos , Reproducibilidad de los ResultadosRESUMEN
The transcellular transport of ingested food ingredients across the intestinal epithelial barrier is an important factor determining bioavailability upon oral intake. This transcellular transport of many chemicals, food ingredients, drugs or toxic compounds over the intestinal epithelium can be highly dependent on the activity of membrane bound ATP binding cassette (ABC) transport proteins, able to export the compounds from the intestinal cells. The present review describes the ABC transporters involved in the efflux of bioactive compounds from the intestinal cells, either to the basolateral blood side, facilitating absorption, or back into the intestinal lumen, reducing bioavailability. The role of the ABC transporters in intestinal transcellular uptake also implies a role for inhibitors of these transporters in modulation of the bioavailability upon oral uptake. The present paper focuses on the role of flavonoids as important modulators or substrates of intestinal ABC transport proteins. Several examples of such an effect of flavonoids are presented. It can be concluded that flavonoid-mediated inhibition of ABC transporters may affect the bioavailability of drugs, bioactive food ingredients and/or food-borne toxic compounds upon oral uptake. All together it appears that the flavonoid-mediated interactions at the level of the intestinal ABC transport proteins may be an important mechanism for unexpected food-drug, food-toxin or food-food interactions. The overview also indicates that future studies should focus on i) in vivo validation of the flavonoid-mediated effects on bioavailability of drugs, toxins and beneficial bioactive food ingredients detected in in vitro models, and on ii) the role of flavonoid phase II metabolism in modulating the activity of the flavonoids to act as ABC transporter inhibitors and/or substrates.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Disponibilidad Biológica , Flavonoides/farmacología , Mucosa Intestinal/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Resistencia a Múltiples Medicamentos , Flavonoides/administración & dosificación , HumanosRESUMEN
The present study describes the effect of different flavonoids on the absorption of the pro-carcinogen PhIP through Caco-2 monolayers and the development of an in silico model describing this process taking into account passive diffusion and active transport of PhIP. Various flavonoids stimulated the apical to basolateral PhIP transport. Using the in silico model for flavone, kaempferol and chrysoeriol, the apparent Ki value for inhibition of the active transport to the apical side was estimated to be below 53 muM and for morin, robinetin and taxifolin between 164 and 268 microM. For myricetin, luteolin, naringenin and quercetin, the apparent Ki values were determined more accurately and amounted to 37.3, 12.2, 11.7 and 5.6 microM respectively. Additional experiments revealed that the apical to basolateral PhIP transport was also increased in the presence of a typical BCRP or MRP inhibitor with apparent Ki values in the same range as those of the flavonoids. This observation together with the fact that flavonoids are known to be inhibitors of MRPs and BCRP, corroborates that inhibition of these apical membrane transporters is involved in the flavonoid-mediated increased apical to basolateral PhIP transport. Based on the apparent Ki values obtained, it is concluded that the flavonols, at the levels present in the regular Western diet, are capable of stimulating the transport of PhIP through Caco-2 monolayers from the apical to the basolateral compartment. This points to flavonoid-mediated stimulation of the bioavailability of PhIP and, thus, a possible adverse effect of these supposed beneficial food ingredients.
Asunto(s)
Carcinógenos/metabolismo , Flavonoides/farmacología , Imidazoles/metabolismo , Absorción Intestinal/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/metabolismo , Acridinas/farmacología , Transporte Biológico Activo/efectos de los fármacos , Células CACO-2 , Permeabilidad de la Membrana Celular/efectos de los fármacos , Difusión , Relación Dosis-Respuesta a Droga , Flavanonas/farmacología , Humanos , Mucosa Intestinal/metabolismo , Cinética , Proteínas de Transporte de Membrana/metabolismo , Modelos Biológicos , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Propionatos/farmacología , Quinolinas/farmacología , Reproducibilidad de los Resultados , Tetrahidroisoquinolinas/farmacologíaRESUMEN
This is the report of the first workshop "Validation of Toxicogenomics-Based Test Systems" held 11-12 December 2003 in Ispra, Italy. The workshop was hosted by the European Centre for the Validation of Alternative Methods (ECVAM) and organized jointly by ECVAM, the U.S. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), and the National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM). The primary aim of the workshop was for participants to discuss and define principles applicable to the validation of toxicogenomics platforms as well as validation of specific toxicologic test methods that incorporate toxicogenomics technologies. The workshop was viewed as an opportunity for initiating a dialogue between technologic experts, regulators, and the principal validation bodies and for identifying those factors to which the validation process would be applicable. It was felt that to do so now, as the technology is evolving and associated challenges are identified, would be a basis for the future validation of the technology when it reaches the appropriate stage. Because of the complexity of the issue, different aspects of the validation of toxicogenomics-based test methods were covered. The three focus areas include a) biologic validation of toxicogenomics-based test methods for regulatory decision making, b) technical and bioinformatics aspects related to validation, and c) validation issues as they relate to regulatory acceptance and use of toxicogenomics-based test methods. In this report we summarize the discussions and describe in detail the recommendations for future direction and priorities.
Asunto(s)
Toxicogenética/legislación & jurisprudencia , Alternativas a las Pruebas en Animales/legislación & jurisprudencia , Biología Computacional , Regulación Gubernamental , Reproducibilidad de los Resultados , Pruebas de Toxicidad/métodosRESUMEN
The effect of the flavonoid myricetin on the transport of the pro-carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) through differentiated Caco-2 monolayers, a model for the intestinal epithelium, is described. Myricetin causes an increase of the transport of PhIP from the apical to the basolateral compartment. This effect was observed at physiologically relevant concentrations of PhIP and myricetin. Cyclosporin A (MRP2 inhibitor) but not PSC833 (P-gp inhibitor) showed a similar effect on PhIP transport. The results indicate that myricetin induces an increased basolateral uptake of the pro-carcinogen PhIP, in part through inhibition of the MRP2 mediated excretion of PhIP from the intestinal cells back to the lumen.
Asunto(s)
Carcinógenos/farmacocinética , Flavonoides/farmacología , Imidazoles/farmacocinética , Transportadoras de Casetes de Unión a ATP/fisiología , Absorción , Células CACO-2 , Humanos , Proteínas de Transporte de Membrana/metabolismo , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , PermeabilidadRESUMEN
Toxicogenomics can facilitate the identification and characterization of toxicity, as illustrated in this review. Toxicogenomics, the application of the functional genomics technologies (transcriptomics, proteomics and metabolomics) in toxicology enables the study of adverse effects of xenobiotic substances in relation to structure and activity of the genome. The advantages and limitations of the different technologies are evaluated, and the prospects for integration of the technologies into a systems biology or systems toxicology approach are discussed. Applications of toxicogenomics in various laboratories around the world show that the crucial steps and sequence of events at the molecular level can be studied to provide detailed insights into mechanisms of toxic action. Toxicogenomics allowed for more sensitive and earlier detection of adverse effects in (animal) toxicity studies. Furthermore, the effects of exposure to mixtures could be studied in more detail. This review argues that in the (near) future, human health risk assessment will truly benefit from toxicogenomics (systems toxicology).
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Proteómica , Biología de Sistemas , Toxicogenética , Transcripción Genética/genética , Animales , Humanos , Medición de RiesgoRESUMEN
This study investigated whether integrated analysis of transcriptomics and metabolomics data increased the sensitivity of detection and provided new insight in the mechanisms of hepatotoxicity. Metabolite levels in plasma or urine were analyzed in relation to changes in hepatic gene expression in rats that received bromobenzene to induce acute hepatic centrilobular necrosis. Bromobenzene-induced lesions were only observed after treatment with the highest of 3 dose levels. Multivariate statistical analysis showed that metabolite profiles of blood plasma were largely different from controls when the rats were treated with bromobenzene, also at doses that did not elicit histopathological changes. Changes in levels of genes and metabolites were related to the degree of necrosis, providing putative novel markers of hepatotoxicity. Levels of endogenous metabolites like alanine, lactate, tyrosine and dimethylglycine differed in plasma from treated and control rats. The metabolite profiles of urine were found to be reflective of the exposure levels. This integrated analysis of hepatic transcriptomics and plasma metabolomics was able to more sensitively detect changes related to hepatotoxicity and discover novel markers. The relation between gene expression and metabolite levels was explored and additional insight in the role of various biological pathways in bromobenzene-induced hepatic necrosis was obtained, including the involvement of apoptosis and changes in glycolysis and amino acid metabolism. The complete Table 2 is available as a supplemental file online at http://taylorandfrancis.metapress.com/openurlasp?genre=journal&issn=0192-6233. To access the file, click on the issue link for 33(4), then select this article. A download option appears at the bottom of this abstract. In order to access the full article online, you must either have an individual subscription or a member subscription accessed through www.toxpath.org.
Asunto(s)
Bromobencenos , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Perfilación de la Expresión Génica , Hígado/efectos de los fármacos , Transcripción Genética/genética , Aminoácidos/sangre , Aminoácidos/orina , Animales , Bromobencenos/farmacocinética , Bromobencenos/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Relación Dosis-Respuesta a Droga , Hígado/metabolismo , Hígado/patología , Masculino , Necrosis , Análisis de Componente Principal , Ratas , Ratas Wistar , Transcripción Genética/efectos de los fármacosRESUMEN
Benzene is an industrial chemical, component of automobile exhaust and cigarette smoke. After hepatic bioactivation benzene induces bone marrow, blood and hepatic toxicity. Using a toxicogenomics approach this study analysed the effects of benzene at three dose levels on gene expression in the liver after 28 daily doses. NMR based metabolomics was used to assess benzene exposure by identification of characteristic benzene metabolite profiles in urine. The 28-day oral exposure to 200 and 800 mg/kg/day but not 10 mg/kg/day benzene-induced hematotoxicity in male Fisher rats. Additionally these upper dose levels slightly reduced body weight and increased relative liver weights. Changes in hepatic gene expression were identified with oligonucleotide microarrays at all dose levels including the 10 mg/kg/day dose level where no toxicity was detected by other methods. The benzene-induced gene expression changes were related to pathways of biotransformation, glutathione synthesis, fatty acid and cholesterol metabolism and others. Some of the effects on gene expression observed here have previously been observed after induction of acute hepatic necrosis with bromobenzene and acetaminophen. In conclusion, changes in hepatic gene expression were found after treatment with benzene both at the toxic and non-toxic doses. The results from this study show that toxicogenomics identified hepatic effects of benzene exposure possibly related to toxicity. The findings aid to interpret the relevance of hepatic gene expression changes in response to exposure to xenobiotics. In addition, the results have the potential to inform on the mechanisms of response to benzene exposure.
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Benceno/toxicidad , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Hígado/efectos de los fármacos , Mutágenos/toxicidad , Animales , Recuento de Células Sanguíneas , Colesterol/metabolismo , Ácidos Grasos/metabolismo , Hemoglobinas/metabolismo , Hígado/metabolismo , Hígado/patología , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Tamaño de los Órganos/efectos de los fármacos , Ratas , Ratas Endogámicas F344 , Bazo/efectos de los fármacos , Bazo/inmunología , Bazo/patología , Timo/efectos de los fármacos , Timo/patología , Factores de Tiempo , UrinálisisRESUMEN
BACKGROUND: Milk products are a potential matrix for fortification with synthetic folic acid or natural 5-methyltetrahydrofolate (5-CH3-H4folate) to enhance the daily folate intake. In milk, folate occurs bound to folate-binding proteins (FBP). Our previous studies with an in vitro gastrointestinal model showed that 70% of the initial FBP content of the milk product was retained in the duodenal lumen. While folic acid remained bound to FBP after gastric passage, 5-CH3-H4folate was mainly present as free folate in the duodenal lumen. AIM OF THE STUDY: To investigate the effect of FBP on the absorption of folic acid and 5-CH3-H4folate from the intestinal lumen. METHODS: The transport of [3H]-folic acid and [14C]-5-CH3-H4folate across enterocytes was studied in the presence or absence of bovine FBP using monolayers of Caco-2 cells grown on semi-permeable inserts in a two-compartment model. The apparent permeability coefficients (P(app)) of folic acid and 5-CH3-H4folate were determined and compared with the permeability of reference compounds for low (mannitol) and high (caffeine) permeability. RESULTS: The transport from the apical to the basolateral side of the Caco-2 cells was higher (P < 0.05) for folic acid (P(app) = 1.7*10(-6) cm/s) than for 5-CH3-H4folate (P(app) = 1.4*10(-6) cm/s) after 2 h incubation to 1 microM folic acid or 5-CH3-H4folate test solutions (pH 7). The permeability of folic acid and 5-CH3-H4folate across Caco-2 monolayers appeared to be higher (P < 0.05) than that of mannitol (P(app) = 0.5*10(-6) cm/s) but lower (P < 0.05) than that of caffeine (P(app) = 34*10(-6) cm/s). The addition of FBP to the medium led to a lower (P < 0.05) intestinal transport and cellular accumulation of folic acid and 5-CH3-H4folate. CONCLUSIONS: Compared to the reference compounds, folic acid and 5-CH3-H4folate showed a moderate permeability across Caco-2 cells, which indicates that folate absorption from the intestinal lumen is not likely to be complete. The intestinal transport of folic acid and 5-CH3-H4folate was found to be dependent on the extent of binding to FBP at the luminal side of the cells.
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Proteínas Portadoras/farmacología , Ácido Fólico/metabolismo , Intestino Delgado/metabolismo , Tetrahidrofolatos/metabolismo , Transporte Biológico/efectos de los fármacos , Células CACO-2 , Proteínas Portadoras/metabolismo , Técnicas de Cultivo de Célula , Cromatografía en Gel , Receptores de Folato Anclados a GPI , Humanos , Absorción Intestinal , Receptores de Superficie Celular/metabolismo , Factores de TiempoRESUMEN
Rats were exposed to three levels of bromobenzene, sampled at 6, 24, and 48 h, and liver gene expression profiles were determined to identify dose and time-related changes. Expression of many genes changed transiently, and dependent on the dose. Few changes were identified after 6 h, but many genes were differentially expressed after 24 h, while after 48 h, only the high dose elicited large effects. Differentially expressed genes were involved in drug metabolism (upregulated GSTs, mEH, NQO1, Mrps, downregulated CYPs, sulfotransferases), oxidative stress (induced HO-1, peroxiredoxin, ferritin), GSH depletion (induced GCS-l, GSTA, GSTM) the acute phase response, and in processes like cholesterol, fatty acid and protein metabolism, and intracellular signaling. Trancriptional regulation via the electrophile and sterol response elements seemed to mediate part of the response to bromobenzene. Recovery of the liver was suggested in response to BB by the altered expression of genes involved in protein synthesis and cytoskeleton rearrangement. Furthermore, after 48 h, rats in the mid dose group showed no toxicity, and gene expression patterns resembled the normal situation. For certain genes (e.g., CYP4A, metallothioneins), intraday variation in expression levels was found, regardless of the treatment. Selected cDNA microarray measurements were confirmed using the specific and sensitive branched DNA signal amplification assay.
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Bromobencenos/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Hígado/efectos de los fármacos , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/sangre , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Glutatión/genética , Glutatión/metabolismo , Hígado/metabolismo , Hígado/patología , Masculino , Análisis por Micromatrices , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Ratas Endogámicas , Factores de Tiempo , Transcripción Genética/efectos de los fármacosRESUMEN
The effect of thiabendazole (TB) on some rat hepatic xenobiotic metabolising enzymes has been investigated. Male Sprague-Dawley rats were fed control diet or diets containing 102-5188 ppm TB for 28 days. As a positive control for induction of hepatic xenobiotic metabolism, rats were also fed diets containing 1457 and 10,155 ppm butylated hydroxytoluene (BHT). Treatment with TB and BHT resulted in dose-dependent increases in relative liver weight. TB was found to be a mixed inducer of cytochrome P450 (CYP) forms in the CYP1A and CYP2B subfamilies. The administration of high doses of TB resulted in the induction of 7-ethoxyresorufin O-deethylase and 7-pentoxyresorufin O-depentylase activities, CYP1A1, CYP1A2, CYP2B1 and CYP2B1/2 mRNA levels and CYP1A2 and CYP2B1/2 apoprotein levels. In contrast, BHT was a CYP2B form inducer, increasing 7-pentoxyresorufin O-depentylase activity, CYP2B1 and CYP2B1/2 mRNA levels and CYP2B1/2 apoprotein levels. Both TB and BHT induced GSH S-transferase activities towards a range of substrates. In addition, TB and BHT markedly induced GSTP1 mRNA levels, but had only a small effect on GSTT1 mRNA levels. In summary, these results demonstrate that TB induces both phase I and II xenobiotic metabolising enzymes in rat liver.