RESUMEN
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.
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Hígado , Proteómica , Animales , Sistema Enzimático del Citocromo P-450/metabolismo , Hepatocitos/metabolismo , Hígado/metabolismo , Espectrometría de Masas , Ratones , Proteínas Quinasas/metabolismoRESUMEN
Activation of the constitutive androstane receptor (CAR) may induce adaptive but also adverse effects in rodent liver, including the induction of drug-metabolizing enzymes, transient hepatocellular proliferation, and promotion of liver tumor growth. Human relevance of CAR-related adverse hepatic effects is controversially debated. Here, we used the chimeric FRG-KO mouse model with livers largely repopulated by human hepatocytes, in order to study human hepatocytes and their response to treatment with the model CAR activator phenobarbital (PB) in vivo. Mice received an intraperitoneal injection with 50 mg/kg body weight PB or saline, and were sacrificed after 72-144 h. Non-repopulated FRG-KO mice were used as additional control. Comprehensive proteomics datasets were generated by merging data obtained by targeted as well as non-targeted proteomics approaches. For the first time, a novel proteomics workflow was established to comparatively analyze the effects of PB on human and murine proteins within one sample. Analysis of merged proteome data sets and bioinformatics data mining revealed comparable responses in murine and human hepatocytes with respect to nuclear receptor activation and induction of xenobiotic metabolism. By contrast, activation of MYC, a key regulator of proliferation, was predicted only for mouse but not human hepatocytes. Analyses of 5-bromo-2'-deoxyuridine incorporation confirmed this finding. In summary, this study for the first time presents a comprehensive proteomic analysis of CAR-dependent effects in human and mouse hepatocytes from humanized FRG-KO mice. The data support the hypothesis that PB does induce adaptive metabolic responses, but not hepatocellular proliferation in human hepatocytes in vivo.
Asunto(s)
Fenobarbital , Proteómica , Animales , Receptor de Androstano Constitutivo , Hepatocitos , Humanos , Hígado , Ratones , Ratones Endogámicos , Fenobarbital/toxicidadRESUMEN
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.
Asunto(s)
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
Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants produced by incomplete combustion of organic matter. They induce their own metabolism by upregulating xenobiotic-metabolizing enzymes such as cytochrome P450 monooxygenase 1A1 (CYP1A1) by activating the aryl hydrocarbon receptor (AHR). However, previous studies showed that individual PAHs may also interact with the constitutive androstane receptor (CAR). Here, we studied ten PAHs, different in carcinogenicity classification, for their potential to activate AHR- and CAR-dependent luciferase reporter genes in human liver cells. The majority of investigated PAHs activated AHR, while non-carcinogenic PAHs tended to activate CAR. We further characterized gene expression, protein abundancies and activities of the AHR targets CYP1A1 and 1A2, and the CAR target CYP2B6 in human HepaRG hepatoma cells. Enzyme induction patterns strongly resembled the profiles obtained at the receptor level, with AHR-activating PAHs inducing CYP1A1/1A2 and CAR-activating PAHs inducing CYP2B6. In summary, this study provides evidence that beside well-known activation of AHR, some PAHs also activate CAR, followed by subsequent expression of respective target genes. Furthermore, we found that an increased PAH ring number is associated with AHR activation as well as the induction of DNA double-strand breaks, whereas smaller PAHs activated CAR but showed no DNA-damaging potential.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Carcinoma Hepatocelular/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias Hepáticas/metabolismo , Hidrocarburos Policíclicos Aromáticos/farmacología , Receptores de Hidrocarburo de Aril/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Xenobióticos/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Receptor de Androstano Constitutivo , Sistema Enzimático del Citocromo P-450/metabolismo , Inducción Enzimática , Células Hep G2 , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Receptores de Hidrocarburo de Aril/genética , Receptores Citoplasmáticos y Nucleares/genética , Activación TranscripcionalRESUMEN
Multitransmembrane proteins are notoriously difficult to analyze. To date, rapid, and cost-efficient detection methods are lacking and only mass spectrometry-based systems allow reliable quantification of these proteins. Here, we present a novel type of sandwich immunoassay that is capable of sensitively detecting multidrug resistance protein 1 (MDR1), a prototypic 12-transmembrane-domains transporter. In a first assay step, complex samples are enzymatically fragmented into peptides as routinely done for mass spectrometry. A proteotypic peptide derived from MDR1 was chosen and antibodies targeting this peptide were used to build a sandwich immunoassay. Validation of the optimized assay showed good sensitivity, reproducibility and it allowed reliable quantification of MDR1; cross-validation by mass spectrometry demonstrated the applicability for routine analyses in clinical and pharmaceutical research. MDR1 was quantified in primary human renal cell carcinoma and corresponding normal tissue and down-regulation or expression loss was found in tumor tissue corroborating its importance in drug resistance and efficacy.
Asunto(s)
Carcinoma de Células Renales/química , Inmunoensayo , Neoplasias Renales/química , Péptidos/química , Subfamilia B de Transportador de Casetes de Unión a ATP/análisis , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Humanos , Neoplasias Renales/metabolismo , Neoplasias Renales/patologíaRESUMEN
The quantification of drug metabolizing enzymes and transporters has recently been revolutionized on the basis of targeted proteomic approaches. Isotope-labeled peptides are used as standards for the quantification of the corresponding proteins in enzymatically fragmented samples. However, hurdles in these approaches are low throughput and tedious sample prefractionation steps prior to mass spectrometry (MS) readout. We have developed an assay platform using sensitive and selective immunoprecipitation coupled with mass spectrometric readout allowing the quantification of proteins directly from whole cell lysates using less than 20,000 cells per analysis. Peptide group-specific antibodies (triple X proteomics antibodies) enable the enrichment of proteotypic peptides sharing a common terminus. These antibodies were employed to establish a MS-based immunoassay panel for the quantification of 14 cytochrome P450 (P450) enzymes and nine transporters. We analyzed the P450 enzyme and transporter levels in genotyped liver tissue homogenates and microsomes, and in samples from a time course induction experiment in human hepatocytes addressing different induction pathways. For the analysis of P450 enzymes and transporters only a minute amount of sample is required and no prefractionation is necessary, thus the assay platform bears the potential to bridge cell culture model experiments and results from whole organ tissue studies.
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Transporte Biológico/fisiología , Sistema Enzimático del Citocromo P-450/metabolismo , Inmunoensayo/métodos , Espectrometría de Masas/métodos , Proteínas de Transporte de Membrana/metabolismo , Línea Celular Tumoral , Células Hep G2 , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , Microsomas Hepáticos/metabolismo , Péptidos/metabolismo , Proteómica/métodosRESUMEN
Consumers are exposed to pesticide residues and other food contaminants via the diet. Both can exert adverse effects on different target organs via the activation of nuclear receptor pathways. Hepatotoxic effects of the widely used triazole fungicide propiconazole (Pi) are generally attributed to the activation of the constitutive androstane receptor (CAR) or the pregnane X receptor (PXR). We now investigated the effects of Pi on the aryl hydrocarbon receptor (AHR) and possible mixture toxicity when Pi is present in combination with BbF, an AHR ligand. In silico docking simulations indicate that Pi can bind to human AHR. Subsequent dual luciferase reporter gene assays in human HepG2 cells showed that Pi activates the AHR in vitro. This concentration-dependent activation was confirmed by real-time RT-PCR analyses of the model AHR target genes CYP1A1 and CYP1A2 in human HepaRG and HepG2 cells. In addition, induction of CYP1A1 protein levels and enzyme activity were recorded. Similarly, increased mRNA expression and enzyme activity of Cyp1a1 and Cyp1a2 was observed in livers of rats treated with Pi for 28 days via the diet. Gene expression analysis in AHR-knockout HepaRG cells showed no induction of CYP1A1 and CYP1A2, whereas gene expression in CAR-, and PXR-knockout cells was induced. Finally, mixture effects of Pi and BbF were analyzed in human cell lines: modeling of concentration-response curves revealed concentration additivity. In conclusion, our results demonstrate that the triazole Pi is an activator of AHR in silico, in vitro and in vivo and causes additive effects with an established AHR ligand.
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Fluorenos/toxicidad , Receptores de Hidrocarburo de Aril/efectos de los fármacos , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Triazoles/toxicidad , Animales , Línea Celular , Simulación por Computador , Receptor de Androstano Constitutivo , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Relación Dosis-Respuesta a Droga , Fluorenos/administración & dosificación , Fungicidas Industriales/administración & dosificación , Fungicidas Industriales/toxicidad , Perfilación de la Expresión Génica/métodos , Técnicas de Inactivación de Genes , Genes Reporteros , Células Hep G2 , Humanos , Ligandos , Hígado/efectos de los fármacos , Simulación del Acoplamiento Molecular , Ratas , Ratas Wistar , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Triazoles/administración & dosificaciónRESUMEN
Many different methods are used for mass-spectrometry-based protein quantification in pharmacokinetics and systems pharmacology. It has not been established to what extent the results from these various methods are comparable. Here, we compared six different mass spectrometry-based proteomics methods by measuring the expression of clinically relevant drug transporters and metabolizing enzymes in human liver. Mean protein concentrations were in general quantified to similar levels by methods using whole tissue lysates. Methods using subcellular membrane fractionation gave incomplete enrichment of the proteins. When the enriched proteins were adjusted to levels in whole tissue lysates, they were on average 4-fold lower than those quantified directly in whole tissue lysates. The differences in protein levels were propagated into differences in predictions of hepatic clearance. In conclusion, caution is needed when comparing and applying quantitative proteomics data obtained with different methods, especially since membrane fractionation is common practice for protein quantification used in drug clearance predictions.
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Espectrometría de Masas/métodos , Proteínas de la Membrana/metabolismo , Proteómica/métodos , Humanos , Hígado/metabolismoRESUMEN
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.
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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
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
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.
Asunto(s)
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.
Asunto(s)
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
Pre-SARS-CoV-2, tuberculosis was the leading cause of death by a single pathogen. Repetitive exposure of Mycobacterium tuberculosis(Mtb) supported the development of multidrug- and extensively drug-resistant strains, demanding novel drugs. Hyperforin, a natural type A polyprenylated polycyclic acylphloroglucinol from St. John's wort, exhibits antidepressant and antibacterial effects also against Mtb. Yet, Hyperforin's instability limits the utility in clinical practice. Here, we present photo- and bench-stable type B PPAPs with enhanced antimycobacterial efficacy. PPAP22 emerged as a lead compound, further improved as the sodium salt PPAP53, drastically enhancing solubility. PPAP53 inhibits the growth of virulent extracellular and intracellular Mtb without harming primary human macrophages. Importantly, PPAP53 is active against drug-resistant strains of Mtb. Furthermore, we analyzed the in vitro properties of PPAP53 in terms of CYP induction and the PXR interaction. Taken together, we introduce type PPAPs as a new class of antimycobacterial compounds, with remarkable antibacterial activity and favorable biophysical properties.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Humanos , Terpenos/farmacología , Antibacterianos/farmacología , Antituberculosos/farmacologíaRESUMEN
Triazole fungicides such as propiconazole (Pi) or tebuconazole (Te) show hepatotoxicity in vivo, e.g., hypertrophy and vacuolization of liver cells following interaction with nuclear receptors such as PXR (pregnane-X-receptor) and CAR (constitutive androstane receptor). Accordingly, azoles affect gene expression associated with these adverse outcomes in vivo but also in human liver cells in vitro. Additionally, genes indicative of liver cholestasis are affected in vivo and in vitro. We therefore analyzed the capability of Pi and Te to cause cholestasis in an adverse outcome pathway (AOP)-driven approach in hepatic cells of human origin in vitro, considering also previous in vivo studies. Bile salt export pump (BSEP) activity assays confirmed that both azoles are weak inhibitors of BSEP. They alternate the expression of various cholestasis-associated target genes and proteins as well as the mitochondrial membrane function. Published in vivo data, however, demonstrate that neither Pi nor Te cause cholestasis in rodent bioassays. This discrepancy can be explained by the in vivo concentrations of both azoles being well below their EC50 for BSEP inhibition. From a regulatory perspective, this illustrates that toxicogenomics and human in vitro models are valuable tools to detect the potential of a substance to cause a specific type of toxicity. To come to a sound regulatory conclusion on the in vivo relevance of such a finding, results will have to be considered in a broader context also including toxicokinetics in a weight-of-evidence approach.
Asunto(s)
Rutas de Resultados Adversos , Colestasis , Fungicidas Industriales , Humanos , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP/genética , Fungicidas Industriales/toxicidad , Azoles/farmacología , Receptores Citoplasmáticos y Nucleares , Triazoles/farmacología , PregnanosRESUMEN
Okadaic acid (OA) is an algae-produced lipophilic marine biotoxin that accumulates in the fatty tissue of filter-feeding shellfish. Ingestion of contaminated shellfish leads to the diarrheic shellfish poisoning syndrome. Furthermore, several other effects of OA like genotoxicity, liver toxicity and tumor-promoting properties have been observed, probably linked to the phosphatase-inhibiting properties of the toxin. It has been shown that at high doses OA can disrupt the physical barrier of the intestinal epithelium. As the intestine and the liver do not only constitute a physical, but also a metabolic barrier against xenobiotic exposure, we here investigated the impact of OA on the expression of cytochrome P450 (CYP) enzymes and transporter proteins in human HepaRG cells liver cells in vitro at non-cytotoxic concentrations. The interplay of OA with known CYP inducers was also studied. Data show that the expression of various xenobiotic-metabolizing CYPs was downregulated after exposure to OA. Moreover, OA was able to counteract the activation of CYPs by their inducers. A number of transporters were also mainly downregulated. Overall, we demonstrate that OA has a significant effect on xenobiotic metabolism barrier in liver cells, highlighting the possibility for interactions of OA exposure with the metabolism of drugs and xenobiotics.
RESUMEN
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.