RESUMEN
Emerging urinary kidney safety biomarkers have been evaluated in recent years and have been shown to be superior to the serum parameters blood urea nitrogen (BUN) and creatinine (sCr) for monitoring kidney injury in the proximal tubule. However, their potential application in differentiating the location of the initial kidney injury (eg, glomerulus vs tubule) has not been fully explored. Here, we assessed the performance of two algorithms that were constructed using either an empirical or a mathematical model to predict the site of kidney injury using a data set consisting of 22 rat kidney toxicity studies with known urine biomarker and histopathologic outcomes. Two kidney safety biomarkers used in both models, kidney injury molecule 1 (KIM-1) and albumin (ALB), were the best performers to differentiate glomerular injury from tubular injury. The performance of algorithms using these two biomarkers against the gold standard of kidney histopathologic examination showed high sensitivity in differentiating the location of the kidney damage to either the glomerulus or the proximal tubules. These data support the exploration of such an approach for use in clinical settings, leveraging urinary biomarker data to aid in the diagnosis of either glomerular or tubular injury where histopathologic assessments are not conducted.
Asunto(s)
Biomarcadores , Glomérulos Renales , Animales , Ratas , Glomérulos Renales/patología , Biomarcadores/orina , Biomarcadores/sangre , Albuminuria , Masculino , Túbulos Renales/patología , Algoritmos , Ratas Sprague-Dawley , Albúminas/metabolismo , Albúminas/análisis , Receptor Celular 1 del Virus de la Hepatitis A/análisis , Lesión Renal Aguda/orina , Lesión Renal Aguda/patología , Moléculas de Adhesión CelularRESUMEN
AIMS: Dicloxacillin is used to treat staphylococcal infections and we have previously shown that dicloxacillin is an inducer of cytochrome P450 enzymes (CYPs). Here, we employed a translational approach to investigate the effect of a treatment with dicloxacillin on warfarin efficacy in Danish registries. Furthermore, we assessed dicloxacillin as an inducer of CYPs in vitro. METHODS: We conducted a register-based study and analysed international normalized ratio (INR) levels in chronic warfarin users before and after short- and long-term use of dicloxacillin (n = 1023) and flucloxacillin (n = 123). Induction of CYPs were investigated in a novel liver model of 3D spheroid primary human hepatocytes at the level of mRNA, and protein and enzyme activity. RESULTS: Short- and long-term dicloxacillin treatments decreased INR levels by -0.65 (95% confidence interval [CI]: -0.57 to -0.74) and -0.76 (95% CI: -0.50 to -1.02), respectively. More than 90% of individuals experienced subtherapeutic INR levels (below 2) after long-term dicloxacillin treatment. Flucloxacillin decreased INR levels by -0.37 (95% CI: -0.14 to -0.60). In 3D spheroid primary human hepatocytes, the maximal induction of CYP3A4 mRNA, protein and enzyme activity by dicloxacillin were 4.9-, 2.9- and 2.4-fold, respectively. Dicloxacillin also induced CYP2C9 mRNA by 1.7-fold. CONCLUSION: Dicloxacillin induces CYPs and reduces the clinical efficacy of warfarin in patients. This effect is substantially exacerbated during long-term treatment with dicloxacillin. The in vitro results corroborated this drug-drug interaction and correlated to the clinical findings. Caution is warranted for warfarin patients that initiate dicloxacillin or flucloxacillin, especially for a long-term treatment of endocarditis.
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Dicloxacilina , Warfarina , Humanos , Warfarina/efectos adversos , Dicloxacilina/farmacología , Anticoagulantes/efectos adversos , Floxacilina/farmacología , Relación Normalizada Internacional , Sistema Enzimático del Citocromo P-450/genética , Hepatocitos , Interacciones FarmacológicasRESUMEN
Periportal and perivenous hepatocytes show zonal heterogeneity in metabolism and signaling. Here, hepatic zonation in mouse liver was analyzed by non-targeted mass spectrometry (MS) and by the antibody-based DigiWest technique, yielding a comprehensive overview of protein expression in periportal and perivenous hepatocytes. Targeted immunoaffinity-based proteomics were used to substantiate findings related to drug metabolism. 165 (MS) and 82 (DigiWest) zonated proteins were identified based on the selected criteria for statistical significance, including 7 (MS) and 43 (DigiWest) proteins not identified as zonated before. New zonated proteins especially comprised kinases and phosphatases related to growth factor-dependent signaling, with mainly periportal localization. Moreover, the mainly perivenous zonation of a large panel of cytochrome P450 enzymes was characterized. DigiWest data were shown to complement the MS results, substantially improving possibilities to bioinformatically identify zonated biological processes. Data mining revealed key regulators and pathways preferentially active in either periportal or perivenous hepatocytes, with ß-catenin signaling and nuclear xeno-sensing receptors as the most prominent perivenous regulators, and several kinase- and G-protein-dependent signaling cascades active mainly in periportal hepatocytes. In summary, the present data substantially broaden our knowledge of hepatic zonation in mouse liver at the protein level.
Asunto(s)
Hígado , Proteómica , Animales , Sistema Enzimático del Citocromo P-450/metabolismo , Hepatocitos/metabolismo , Hígado/metabolismo , Espectrometría de Masas , Ratones , Proteínas Quinasas/metabolismoRESUMEN
The PI3-kinase/AKT/mTOR pathway plays a central role in cancer signaling. While p110α is the catalytic α-subunit of PI3-kinase and a major drug target, PTEN is the main negative regulator of the PI3-kinase/AKT/mTOR pathway. PTEN is often down-regulated in cancer, and there are conflicting data on PTEN's role as breast cancer biomarker. PTEN and p110α protein expression in tumors is commonly analyzed by immunohistochemistry, which suffers from poor multiplexing capacity, poor standardization, and antibody crossreactivity, and which provides only semi-quantitative data. Here, we present an automated, and standardized immuno-matrix-assisted laser desorption/ionization mass spectrometry (iMALDI) assay that allows precise and multiplexed quantitation of PTEN and p110α concentrations, without the limitations of immunohistochemistry. Our iMALDI assay only requires a low-cost benchtop MALDI-TOF mass spectrometer, which simplifies clinical translation. We validated our assay's precision and accuracy, with simultaneous enrichment of both target proteins not significantly affecting the precision and accuracy of the quantitation when compared to the PTEN- and p110α-singleplex iMALDI assays (<15% difference). The multiplexed assay's linear range is from 0.6-20 fmol with accuracies of 90-112% for both target proteins, and the assay is free of matrix-related interferences. The inter-day reproducibility over 5-days was high, with an overall CV of 9%. PTEN and p110α protein concentrations can be quantified down to 1.4 fmol and 0.6 fmol per 10 µg of total tumor protein, respectively, in various tumor tissue samples, including fresh-frozen breast tumors and colorectal cancer liver metastases, and patient-derived xenograft (PDX) tumors.
Asunto(s)
Biomarcadores de Tumor , Neoplasias de la Mama , Línea Celular Tumoral , Femenino , Humanos , Rayos Láser , Proteínas de Neoplasias , Fosfohidrolasa PTEN , Reproducibilidad de los Resultados , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Most drugs and xenobiotics are metabolized in the liver. Amongst others, different cytochrome P450 (CYP) enzymes catalyze the metabolic conversion of foreign compounds, and various transport proteins are engaged in the excretion of metabolites from the hepatocytes. Inter-species and inter-individual differences in the hepatic levels and activities of drug-metabolizing enzymes and transporters result from genetic as well as from environmental factors, and play a decisive role in determining the pharmacokinetic properties of a compound in a given test system. To allow for a meaningful comparison of results from metabolism studies, it is, therefore, of utmost importance to know about the specific metabolic properties of the test systems, especially about the levels of metabolic enzymes such as the CYPs. Using a targeted proteomics approach, we, therefore, compared the hepatic levels of important CYP enzymes and transporters in different experimental systems in vivo and in vitro, namely Wistar rats, C57/Bl6 mice, mice humanized for the two xeno-sensing receptors PXR (pregnane-X-receptor) and CAR (constitutive androstane receptor), mice with human hepatocyte-repopulated livers, human HepaRG hepatocarcinoma cells, primary human hepatocytes, and human liver biopsies. In addition, the effects of xenobiotic inducers of drug metabolism on CYP enzymes and transporters were analyzed in selected systems. This study for the first time presents a comprehensive overview of similarities and differences in important drug metabolism-related proteins among the different experimental models.
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Sistema Enzimático del Citocromo P-450/metabolismo , Hígado/enzimología , Proteínas de Transporte de Membrana/metabolismo , Preparaciones Farmacéuticas/metabolismo , Xenobióticos/metabolismo , Animales , Transporte Biológico , Biotransformación , Línea Celular , Receptor de Androstano Constitutivo , Humanos , Isoenzimas , Ratones Endogámicos C57BL , Receptor X de Pregnano/metabolismo , Ratas Wistar , Receptores Citoplasmáticos y Nucleares/metabolismo , Especificidad de la Especie , Especificidad por SustratoRESUMEN
Xenobiotica-metabolizing enzyme (XME) induction is a relevant biological/biochemical process vital to understanding the toxicological profile of xenobiotics. Early recognition of XME induction potential of compounds under development is therefore important, yet its determination by traditional XME activity measurements is time consuming and cost intensive. A proof-of-principle study was therefore designed due to the advent of faster and less cost-intensive methods for determination of enzyme protein and transcript levels to determine whether two such methods may substitute for traditional measurement of XME activity determinations. The results of the study show that determination of enzyme protein levels by peptide group-specific immunoaffinity enrichment/MS and/or determination of gene expression by NanoString nCounter may serve as substitutes for traditional evaluation methodology and/or as an early predictor of potential changes in liver enzymes. In this study, changes of XME activity by the known standard XME inducers phenobarbital, beta-naphthoflavone and Aroclor 1254 were demonstrated by these two methods. To investigate the applicability of these methods to demonstrate XME-inducing activity of an unknown, TS was also examined and found to be an XME inducer. More specifically, TS was found to be a phenobarbital-type inducer (likely mediated by CAR rather than PXR as nuclear receptor), but not due to Ah receptor-mediated or antioxidant response element-mediated beta-naphthoflavone-type induction. The results for TS were confirmed via enzymatic activity measurements. The results of the present study demonstrate the potential applicability of NanoString nCounter mRNA quantitation and peptide group-specific immunoaffinity enrichment/MS protein quantitation for predicting compounds under development to be inducers of liver XME activity.
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Inductores de las Enzimas del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/biosíntesis , Perfilación de la Expresión Génica , Inmunoensayo , Hígado/efectos de los fármacos , Nanotecnología , Transcriptoma , Xenobióticos/metabolismo , Animales , Biotransformación , Inductores de las Enzimas del Citocromo P-450/toxicidad , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/inmunología , Inducción Enzimática , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Hígado/enzimología , Masculino , Prueba de Estudio Conceptual , Ratas Wistar , Reproducibilidad de los Resultados , Especificidad por Sustrato , Toxicocinética , Flujo de Trabajo , Xenobióticos/toxicidadRESUMEN
Iron oxide nanoparticles are used in various industrial fields, as a tool in biomedicine as well as in food colorants, and can therefore reach human metabolism via oral uptake or injection. However, their effects on the human body, especially the liver as one of the first target organs is still under elucidation. Here, we studied the influence of different representative iron oxide materials on xenobiotic metabolism of HepaRG cells. These included four iron oxide nanoparticles, one commercially available yellow food pigment (E172), and non-particulate ionic control FeSO4. The nanoparticles had different chemical and crystalline structures and differed in size and shape and were used at a concentration of 50 µg Fe/mL. We found that various CYP enzymes were downregulated by some but not all iron oxide nanoparticles, with the Fe3O4-particle, both γ-Fe2O3-particles, and FeSO4 exhibiting the strongest effects, the yellow food pigment E172 showing a minor effect and an α-Fe2O3 nanoparticle leading to almost no inhibition of phase I machinery. The downregulation was seen at the mRNA, protein expression, and activity levels. Thereby, no dependency on the size or chemical structure was found. This underlines the difficulty of the grouping of nanomaterials regarding their physiological impact, suggesting that every iron oxide nanoparticle species needs to be evaluated in a case-by-case approach.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Hepatocitos/efectos de los fármacos , Nanopartículas Magnéticas de Óxido de Hierro/toxicidad , Xenobióticos/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/efectos de los fármacos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Biotransformación , Receptor de Androstano Constitutivo , Sistema Enzimático del Citocromo P-450/genética , Regulación hacia Abajo , Regulación Enzimológica de la Expresión Génica , Células Hep G2 , Hepatocitos/enzimología , Humanos , Isoenzimas , Estructura Molecular , Tamaño de la Partícula , Receptor X de Pregnano/efectos de los fármacos , Receptor X de Pregnano/genética , Receptor X de Pregnano/metabolismo , Receptores de Hidrocarburo de Aril/efectos de los fármacos , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Especificidad por Sustrato , Xenobióticos/farmacologíaRESUMEN
Adenosine diphosphate (ADP) enhances platelet activation by virtually any other stimulant to complete aggregation. It binds specifically to the G-protein-coupled membrane receptors P2Y1 and P2Y12, stimulating intracellular signaling cascades, leading to integrin αIIbß3 activation, a process antagonized by endothelial prostacyclin. P2Y12 inhibitors are among the most successful antiplatelet drugs, however, show remarkable variability in efficacy. We reasoned whether a more detailed molecular understanding of ADP-induced protein phosphorylation could identify (1) critical hubs in platelet signaling toward aggregation and (2) novel molecular targets for antiplatelet treatment strategies. We applied quantitative temporal phosphoproteomics to study ADP-mediated signaling at unprecedented molecular resolution. Furthermore, to mimic the antagonistic efficacy of endothelial-derived prostacyclin, we determined how Iloprost reverses ADP-mediated signaling events. We provide temporal profiles of 4797 phosphopeptides, 608 of which showed significant regulation. Regulated proteins are implicated in well-known activating functions such as degranulation and cytoskeletal reorganization, but also in less well-understood pathways, involving ubiquitin ligases and GTPase exchange factors/GTPase-activating proteins (GEF/GAP). Our data demonstrate that ADP-triggered phosphorylation occurs predominantly within the first 10 seconds, with many short rather than sustained changes. For a set of phosphorylation sites (eg, PDE3ASer312, CALDAG-GEFISer587, ENSASer109), we demonstrate an inverse regulation by ADP and Iloprost, suggesting that these are central modulators of platelet homeostasis. This study demonstrates an extensive spectrum of human platelet protein phosphorylation in response to ADP and Iloprost, which inversely overlap and represent major activating and inhibitory pathways.
Asunto(s)
Adenosina Difosfato/metabolismo , Plaquetas/metabolismo , Activación Plaquetaria/fisiología , Transducción de Señal/fisiología , Plaquetas/efectos de los fármacos , Western Blotting , Humanos , Iloprost/farmacología , Fosforilación , Activación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Proteómica/métodosRESUMEN
The agricultural fungicides cyproconazole and prochloraz exhibit hepatotoxicity in rodent studies and are tumorigenic following chronic exposure. Both substances are suspected to act via a CAR (constitutive androstane receptor)/PXR (pregnane-X-receptor)-dependent mechanism. Human relevance of these findings is under debate. A 28-day toxicity study was conducted in mice with humanized CAR and PXR (hCAR/hPXR) with two dose levels (50 or 500 ppm) of both substances, using the model CAR activator phenobarbital as a reference. Results were compared to wild-type mice. A treatment-related increase in liver weights was observed for all three substances at least at the high-dose level. Changes in the expression of classic CAR/PXR target genes such as Cyp2b10 were induced by cyproconazole and phenobarbital in both genotypes, while prochloraz treatment resulted in gene expression changes indicative of additional aryl hydrocarbon receptor activation, e.g. by up-regulation of Cyp1a1 expression. Cyproconazole-induced effects on CAR-dependent gene expression, liver weight, and hepatic lipid accumulation were more prominent in wild-type mice, where significant genotype differences were observed at the high-dose level. Moreover, high-dose cyproconazole-treated mice from the wild-type group responded with a marked increase in hepatocellular proliferation, while hCAR/hPXR mice did not. In conclusion, our data demonstrate that cyproconazole and PB induce CAR/PXR downstream effects in hepatocytes in vivo via both, the murine and human receptors. At high doses of cyproconazole, however, the responses were clearly more pronounced in wild-type mice, indicating increased sensitivity of rodents to CAR agonist-induced effects in hepatocytes.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Fungicidas Industriales/toxicidad , Imidazoles/toxicidad , Triazoles/toxicidad , Animales , Receptor de Androstano Constitutivo , Relación Dosis-Respuesta a Droga , Fungicidas Industriales/administración & dosificación , Regulación de la Expresión Génica/genética , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Humanos , Imidazoles/administración & dosificación , Masculino , Ratones , Ratones Endogámicos C57BL , Fenobarbital/farmacología , Receptor X de Pregnano , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Triazoles/administración & dosificación , Regulación hacia Arriba/efectos de los fármacosRESUMEN
DCs are professional APCs playing a crucial role in the initiation of T-cell responses to combat infection. However, systemic bacterial infection with various pathogens leads to DC-depletion in humans and mice. The mechanisms of pathogen-induced DC-depletion remain poorly understood. Previously, we showed that mice infected with Yersinia enterocolitica (Ye) had impaired de novo DC-development, one reason for DC-depletion. Here, we extend these studies to gain insight into the molecular mechanisms of DC-depletion and the impact of different bacteria on DC-development. We show that the number of bone marrow (BM) hematopoietic progenitors committed to the DC lineage is reduced following systemic infection with different Gram-positive and Gram-negative bacteria. This is associated with a TLR4- and IFN-γ-signaling dependent increase of committed monocyte progenitors in the BM and mature monocytes in the spleen upon Ye-infection. Adoptive transfer experiments revealed that infection-induced monopoiesis occurs at the expense of DC-development. Our data provide evidence for a general response of hematopoietic progenitors upon systemic bacterial infections to enhance monocyte production, thereby increasing the availability of innate immune cells for pathogen control, whereas impaired DC-development leads to DC-depletion, possibly driving transient immunosuppression in bacterial sepsis.
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Diferenciación Celular/inmunología , Células Dendríticas/inmunología , Inmunidad Innata , Mielopoyesis/inmunología , Yersiniosis/inmunología , Yersinia enterocolitica/inmunología , Animales , Células Dendríticas/patología , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/patología , Interferón gamma/inmunología , Ratones , Ratones Noqueados , Transducción de Señal/inmunología , Receptor Toll-Like 4/inmunología , Yersiniosis/patologíaRESUMEN
Blood plasma is a valuable source of potential biomarkers. However, its complexity and the huge dynamic concentration range of its constituents complicate its analysis. To tackle this problem, an immunoprecipitation strategy was employed using antibodies directed against short terminal epitope tags (triple X proteomics antibodies), which allow the enrichment of groups of signature peptides derived from trypsin-digested plasma. Isolated signature peptides are subsequently detected using MALDI-TOF/TOF mass spectrometry. Sensitivity of the immunoaffinity approach was, however, compromised by the presence of contaminant peaks derived from the peptides of nontargeted high abundant proteins. A closer analysis of the enrichment strategy revealed nonspecific peptide binding to the solid phase affinity matrix as the major source of the contaminating peptides. We therefore implemented a sucrose density gradient ultracentrifugation separation step into the procedure. This yielded a 99% depletion of contaminating peptides from a sucrose fraction containing 70% of the peptide-antibody complexes and enabled the detection of the previously undetected low abundance protein filamin-A. Assessment of this novel approach using 15 different triple X proteomics antibodies demonstrated a more consistent detection of a greater number of targeted peptides and a significant reduction in the intensity of nonspecific peptides. Ultracentrifugation coupled with immunoaffinity MS approaches presents a powerful tool for multiplexed plasma protein analysis without the requirement for demanding liquid chromatography separation techniques.
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Proteínas Sanguíneas/análisis , Péptidos/análisis , Proteómica/métodos , Secuencia de Aminoácidos , Anticuerpos/química , Complejo Antígeno-Anticuerpo/química , Biomarcadores/análisis , Proteínas Sanguíneas/química , Centrifugación por Gradiente de Densidad , Proteínas Contráctiles/análisis , Filaminas , Humanos , Inmunoprecipitación/métodos , Proteínas de Microfilamentos/análisis , Datos de Secuencia Molecular , Proteolisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Coloración y Etiquetado , Sacarosa , Tripsina , Ultracentrifugación/métodosRESUMEN
BACKGROUND: and Purpose: Chemotherapy-induced peripheral neuropathy (CIPN) constitutes a significant health problem due to the increasing prevalence and lack of therapies for treatment and prevention. While pivotal for routine cancer treatment, paclitaxel and vincristine frequently cause CIPN and impact the quality of life among cancer patients and survivors. Here, we investigate molecular mechanisms and drug transport in CIPN. EXPERIMENTAL APPROACH: Human sensory neurons were derived from induced pluripotent stem cells (iPSC-SNs), which were characterized using flow cytometry and immunolabeling. These iPSC-SNs were exposed to different concentrations of the two microtubule-targeting agents, paclitaxel and vincristine, with and without pre-exposure to inhibitors and inducers of efflux transporters. Neuronal networks were quantified via fluorescent staining against sensory neuron markers. Transcriptional effects of the chemotherapeutics were examined using quantitative polymerase chain reactions (qPCR). KEY RESULTS: Paclitaxel exposure resulted in axonal retraction and thickening, while vincristine caused fragmentation and abolishment of axons. Both agents increased the mRNA expression of the pain receptor, transient receptor potential vanilloid (TRPV1), and highly induced neuronal damage, as measured by activating transcription factor 3 (ATF3) mRNA. iPSC-SNs express the efflux transporters, P-glycoprotein (P-gp, encoded by ABCB1) and multidrug resistance-associated protein 1 (MPR1, encoded by ABCC1). Modulation of efflux transporters indicate that P-gp and MRP1 play a role in modulating neuronal accumulation and neurotoxicity in preliminary experiments. CONCLUSION: and Implications: iPSC-SNs are a valuable and robust model to study the role of efflux transporters and other mechanistic targets in CIPN. Efflux transporters may play a role in CIPN pathogenesis as they regulate the disposition of chemotherapy to the peripheral nervous system, and they may present potential therapeutic targets for CIPN.
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Células Madre Pluripotentes Inducidas , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Paclitaxel , Enfermedades del Sistema Nervioso Periférico , Células Receptoras Sensoriales , Vincristina , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Humanos , Paclitaxel/toxicidad , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Enfermedades del Sistema Nervioso Periférico/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Antineoplásicos/efectos adversos , Antineoplásicos/toxicidad , Canales Catiónicos TRPV/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Células CultivadasRESUMEN
Cholangiocarcinomas (CCAs) are cancers originated in the biliary tree, which are characterized by their high mortality and marked chemoresistance, partly due to the activity of ATP-binding cassette (ABC) export pumps, whose inhibition has been proposed as a strategy for enhancing the response to chemotherapy. We have previously shown that ß-caryophyllene oxide (CRYO) acts as a chemosensitizer in hepatocellular carcinoma by inhibiting ABCB1, MRP1, and MRP2. Here, we have evaluated the usefulness of CRYO in inhibiting BCRP and improving the response of CCA to antitumor drugs. The TCGA-CHOL cohort (n = 36) was used for in silico analysis. BCRP expression (mRNA and protein) was assayed in samples from intrahepatic (iCCA) and extrahepatic (eCCA) tumors (n = 50) and CCA-derived cells (EGI-1 and TFK-1). In these cells, BCRP-dependent mitoxantrone transport was determined by flow cytometry. At non-toxic concentrations, CRYO inhibited BCRP function, which enhanced the cytostatic effect of drugs used in the treatment of CCA. The BCRP ability to confer resistance to a panel of antitumor drugs was determined in Chinese hamster ovary (CHO) cells with stable BCRP expression. At non-toxic concentrations, CRYO markedly reduced BCRP-induced resistance to known substrate drugs (mitoxantrone and SN-38) and cisplatin, gemcitabine, sorafenib, and 5-FU but not oxaliplatin. Neither CRYO nor cisplatin alone significantly affected the growth of BCRP-expressing tumors subcutaneously implanted in immunodeficient mice. In contrast, intratumor drug content was enhanced when administered together, and tumor growth was inhibited. In sum, the combined treatment of drugs exported by BCRP with CRYO can improve the response to chemotherapy in CCA patients.
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Antineoplásicos , Carcinoma Hepatocelular , Colangiocarcinoma , Neoplasias Hepáticas , Cricetinae , Humanos , Ratones , Animales , Cisplatino/farmacología , Mitoxantrona/farmacología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Células CHO , Resistencia a Antineoplásicos , Transportadoras de Casetes de Unión a ATP , Proteínas de Neoplasias/metabolismo , Cricetulus , Antineoplásicos/farmacología , Colangiocarcinoma/tratamiento farmacológico , Línea Celular TumoralRESUMEN
ß-catenin plays multiple roles in the canonical Wnt signaling pathway and in cell-cell adhesion complexes. In addition, ß-catenin is a proto-oncogene and activating ß-catenin mutations are relevant in the genesis of colorectal, hepatocellular and other common cancers. Different functions of ß-catenin as transcriptional co-activator or cell adhesion molecule are orchestrated by changes in concentration and phosphorylation as well as its ability to complex with proteins such as cadherins or transcription factors. Detailed quantitative and time-resolved analysis of ß-catenin, based on the evaluation of the changes in the Wnt pathway, enable greater insights into health- and disease-related ß-catenin function. The present paper describes a novel suspension bead array assay panel for ß-catenin, which requires minimal amounts of sample and is able to relatively quantify total ß-catenin, the extent of phosphorylation at multiple sites and the ratio of complexed and free ß-catenin. This is the first study to combine three biochemical methods--sandwich immunoassay, co-immunoprecipitation, and protein-protein interaction assay--in one suspension bead assay panel. The assay was used to measure changes in the concentration of eight different ß-catenin forms in HEK293 cells in a time-resolved manner. In contrast to the general consensus, our study demonstrates an increase in ß-catenin phosphorylated at Ser-45 upon treatment of cells with rWnt3a or a GSK3 inhibition; we also link C-terminal phosphorylation of ß-catenin on Ser-552 and Ser-675 with canonical Wnt signaling.
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Procesamiento Proteico-Postraduccional , beta Catenina/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Genes Reporteros , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Células HEK293 , Humanos , Proteínas Inmovilizadas/química , Inmunoprecipitación/métodos , Luciferasas/biosíntesis , Luciferasas/genética , Microesferas , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proto-Oncogenes Mas , Reproducibilidad de los Resultados , Transducción de Señal , Proteínas Wnt/farmacología , Proteínas Wnt/fisiología , Proteína Wnt3 , beta Catenina/químicaRESUMEN
Primary human hepatocytes (PHHs) have been the gold standard in vitro model for the human liver and are crucial to predict hepatic drug-drug interactions. The aim of this work was to assess the utility of 3D spheroid PHHs to study induction of important cytochrome P450 (CYP) enzymes and drug transporters. The 3D spheroid PHHs from three different donors were treated for 4 days with rifampicin, dicloxacillin, flucloxacillin, phenobarbital, carbamazepine, efavirenz, omeprazole, or ß-naphthoflavone. Induction of CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, and transporters P-glycoprotein (P-gp)/ABCB1, multidrug resistance-associated protein 2 (MRP2)/ABCC2, ABCG2, organic cation transporter 1 (OCT1)/SLC22A1, SLC22A7, SLCO1B1, and SLCO1B3 were evaluated at mRNA and protein levels. Enzyme activity of CYP3A4, CYP2B6, CYP2C19, and CYP2D6 were also assessed. Induction of CYP3A4 protein and mRNA correlated well for all donors and compounds and had a maximal induction of five- to sixfold for rifampicin, which closely correlates to induction observed in clinical studies. Rifampicin induced the mRNA of CYP2B6 and CYP2C8 by 9- and 12-fold, whereas the protein levels of these CYPs reached 2- and 3-fold induction, respectively. Rifampicin induced CYP2C9 protein by 1.4-fold, whereas the induction of CYP2C9 mRNA was over 2-fold in all donors. Rifampicin induced ABCB1, ABCC2, and ABCG2 by 2-fold. In conclusion, 3D spheroid PHHs is a valid model to investigate mRNA and protein induction of hepatic drug-metabolizing enzymes and transporters, and this model provides a solid basis to study induction of CYPs and transporters, which translates to clinical relevance.
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Citocromo P-450 CYP3A , Rifampin , Humanos , Citocromo P-450 CYP2C8/metabolismo , Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP3A/metabolismo , Citocromo P-450 CYP2B6/metabolismo , Rifampin/farmacología , Rifampin/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP2C9/metabolismo , Células Cultivadas , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Hepatocitos/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Proteínas Portadoras/metabolismo , ARN Mensajero/metabolismo , Transportador 1 de Anión Orgánico Específico del Hígado/metabolismoRESUMEN
Flucloxacillin is a widely used antibiotic. It is an agonist to the nuclear receptor PXR that regulates the expression of cytochrome P450 (CYP) enzymes. Treatment with flucloxacillin reduces warfarin efficacy and plasma concentrations of tacrolimus, voriconazole, and repaglinide. We conducted a translational study to investigate if flucloxacillin induces CYP enzymes. We also investigated if flucloxacillin induces its own metabolism as an autoinducer. We performed a randomized, unblinded, two-period, cross-over, clinical pharmacokinetic cocktail study. Twelve healthy adults completed the study. They ingested 1 g flucloxacillin 3 times daily for 31 days, and we assessed the full pharmacokinetics of the Basel cocktail drugs on days 0, 10, and 28, and plasma concentrations of flucloxacillin on days 0, 9, and 27. The 3D spheroid of primary human hepatocytes (PHHs) were exposed to flucloxacillin (concentration range: 0.15-250 µM) for 96 hours. Induction of mRNA expression, protein abundance, and enzyme activity of CYP enzymes were assessed. Flucloxacillin treatment reduced the metabolic ratio of midazolam (CYP3A4), (geometric mean ratio (GMR) 10 days (95% confidence interval (CI)): 0.75 (0.64-0.89)) and (GMR 28 days (95% CI): 0.72 (0.62-0.85)). Plasma concentrations of flucloxacillin did not change during 27 days of treatment. Flucloxacillin caused concentration-dependent induction of CYP3A4 and CYP2B6 (mRNA, protein, and activity), CYP2C9 (mRNA and protein), CYP2C19 (mRNA and activity), and CYP2D6 (activity) in 3D spheroid PHH. In conclusion, flucloxacillin is a weak inducer of CYP3A4, which may lead to clinically relevant drug-drug interactions for some narrow therapeutic range drugs that are substrates of CYP3A4.
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Citocromo P-450 CYP3A , Floxacilina , Humanos , Adulto , Citocromo P-450 CYP3A/genética , Floxacilina/farmacología , Sistema Enzimático del Citocromo P-450/metabolismo , Interacciones Farmacológicas , Hepatocitos/metabolismo , ARN MensajeroRESUMEN
BACKGROUND: The Wnt/ß-catenin signalling is aberrantly activated in primary B cell chronic lymphocytic leukaemia (CLL). Epigenetic silencing of pathway inhibitor genes may be a mechanism for its activation. In this study, we investigated systematically and quantitatively the methylation status of 12 Wnt/ß-catenin pathway inhibitor genes - CDH1, DACT1, DKK1, DKK2, DKK3, DKK4, SFRP1, SFRP2, SFRP3, SFRP4, SFRP5 and WIF1 - in the cell lines EHEB and MEC-1 as well as patient samples. METHODS: Quantification of DNA methylation was performed by means of bisulphite pyrosequencing and confirmed by bisulphite Sanger sequencing. Gene expression was analysed by qPCR using GAPDH as internal control. E-cadherin and ß-catenin protein quantification was carried out by microsphere-based immunoassays. Methylation differences observed between the patient and control groups were tested using generalised least squares models. RESULTS: For 10 genes, a higher methylation level was observed in tumour material. Only DKK4 exhibited similarly high methylation levels in both tumour and normal specimens, while DACT1 was always essentially unmethylated. However, also for these inhibitors, treatment of cells with the demethylating agent 5-aza-2´-deoxycytidine resulted in an induction of their expression, as shown by quantitative PCR, suggesting an indirect epigenetic control of activity. While the degree of demethylation and its transcriptional consequences differed between the genes, there was an overall high correlation of demethylation and increased activity. Protein expression studies revealed that no constitutive Wnt/ß-catenin signalling occurred in the cell lines, which is in discrepancy with results from primary CLL. However, treatment with 5-aza-2´-deoxycytidine caused accumulation of ß-catenin. Simultaneously, E-cadherin expression was strongly induced, leading to the formation of a complex with ß-catenin and thus demonstrating its epigenetically regulated inhibition effect. CONCLUSIONS: The results suggest an epigenetic silencing mechanism of the Wnt/ß-catenin pathway inhibitor genes in CLL. Hypermethylation and silencing of functionally related genes may not be completely stochastic but result from the tumour epigenome reprogramming orchestrated by Polycomb-group repressive complexes. The data are of interest in the context of epigenetic-based therapy.
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Epigénesis Genética , Silenciador del Gen , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/metabolismo , Transducción de Señal , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Antimetabolitos Antineoplásicos/farmacología , Azacitidina/farmacología , Cadherinas/metabolismo , Línea Celular Tumoral , Mapeo Cromosómico , Islas de CpG , Metilación de ADN , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Unión ProteicaRESUMEN
SCOPE: 1,2-unsaturated pyrrolizidine alkaloids (PAs) are secondary plant metabolites that are found in many plant species throughout the world. They are of concern for risk assessment as consumption of contaminated foodstuff can cause severe liver damage. Of late, transporter-mediated uptake and transport has advanced as a vital determinant of PA toxicity. In this study, the authors investigate a transporter-mediated uptake of PAs and its implications in PA toxicity. METHODS AND RESULTS: We show that transporter expression levels are significantly affected by treatment with the PAs senecionine (Sc) and retrorsine (Re) in the human hepatoma cell line HepaRG. Furthermore, the specific contribution to PA uptake of the two transporters Na+ /taurocholate co-transporting polypeptide (SLC10A1) and organic cation transporter I (SLC22A1), both belonging to the heterogeneous solute carrier super family, is investigated by means of a siRNA-mediated knockdown approach. Knockdown of both uptake transporters result in reduced uptake of Re and Sc in a time-dependent manner and attenuated PA-mediated cytotoxic effects in HepaRG cells. CONCLUSION: Our results confirm previous findings of active transport mechanisms of PAs into hepatocytes and highlight the importance of toxicokinetic studies for the risk assessment of PAs.
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Enfermedad Hepática Inducida por Sustancias y Drogas , Alcaloides de Pirrolicidina , Cationes/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Hepatocitos , Humanos , Péptidos/metabolismo , Alcaloides de Pirrolicidina/toxicidad , Ácido Taurocólico/metabolismoRESUMEN
BACKGROUND: Platelet glycoprotein VI (pGPVI) expression is increased in acute coronary syndrome (ACS), reflecting platelet activation. There is no reliable method available to measure pGPVI. Our aim was to develop a bead-based sandwich immunoassay to measure soluble GPVI (sGPVI). METHODS: Based on antibodies for sGPVI developed earlier, we established and validated a bead-based sandwich immunoassay in 2438 consecutive patients with stable angina pectoris (SAP; n = 1371), non-ST-elevation myocardial infarction (NSTEMI; n = 724), and ST-elevation MI (STEMI; n = 343). In a subgroup (n = 1011), we measured surface expression of pGPVI using flow cytometry. RESULTS: The assay revealed a working range of 8-500 ng/L. Intra- and interassay imprecision was <7% and <14%, respectively. Patients with NSTEMI and STEMI showed significantly lower mean sGPVI concentrations than patients with SAP [mean (SD), 8.4 (3.6) µg/L and 8.6 (4.1) µg/L vs 9.8 (4.8) µg/L; P = 0.002], whereas subgroup analysis revealed significantly enhanced pGPVI in NSTEMI (n = 276) and STEMI (n = 80) patients compared with SAP (n = 655) [mean fluorescence intensity (SD), 21.2 (8.1) and 19.8 (6.8) vs 18.5 (7.7); P = 0.002 and P = 0.018]. pGPVI and sGPVI were inversely correlated (r = -0.076; P = 0.023). Area under the ROC curve was 0.716, 95% CI 0.681-0.751, for sGPVI, distinguishing patients with SAP from those with ACS, and was superior (P = 0.044) to the curve of subgroup analysis for pGPVI (0.624, 95% CI 0.586-0.662). sGPVI (P = 0.023) and pGPVI (P = 0.028) had better association with the development of ACS than troponin I (P = 0.055) in the very early stage of disease, based on logistic regression analysis. CONCLUSIONS: This sandwich immunoassay reliably measures sGPVI and may help to identify patients with ACS earlier than other laboratory markers.
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Enfermedad de la Arteria Coronaria/diagnóstico , Glicoproteínas de Membrana Plaquetaria/análisis , Síndrome Coronario Agudo/sangre , Síndrome Coronario Agudo/diagnóstico , Anciano , Angina de Pecho/sangre , Angina de Pecho/diagnóstico , Biomarcadores/sangre , Enfermedad de la Arteria Coronaria/sangre , Femenino , Humanos , Inmunoensayo/métodos , Masculino , Infarto del Miocardio/sangre , Infarto del Miocardio/diagnóstico , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , SolubilidadRESUMEN
Toxicological effects of chemicals are mostly tested individually. However, consumers encounter exposure to complex mixtures, for example multiple pesticide residues, by consuming food such as crops, fruits or vegetables. Currently, more than 450 active substances are approved in the European Union, and there is little data on effects after combined exposure to several pesticides. Toxicological animal studies would increase enormously, if pesticide combinations had to be analyzed in vivo. Therefore, in vitro methods addressing this issue are needed. We have developed 32 immunoaffinity-based mass spectrometry assays to investigate the impact of hepatotoxic active substances on liver proteins in human HepaRG cells. Five compounds were selected based on their (dis)similar capability to modulate protein levels, and on their combined use in commercially available formulations. Four binary mixtures were prepared from these five substances and tested in different concentrations over three time points. We applied a novel statistical method to describe deviations from additivity and to detect antagonistic and synergistic effects. The results regarding the abundance of hepatotoxicity-related proteins showed additive behavior for 1323 out of 1427 endpoints tested, while 104 combinatorial effects deviating from additivity, such as antagonism or synergism were observed.