RESUMEN
As the conventional approach to assess the potential of a chemical to cause cancer in humans still includes the 2-year rodent carcinogenicity bioassay, development of alternative methodologies is needed. In the present study, the transcriptomics responses following exposure to genotoxic (GTX) and non-genotoxic (NGTX) hepatocarcinogens and non-carcinogens (NC) in five liver-based in vitro models, namely conventional and epigenetically stabilized cultures of primary rat hepatocytes, the human hepatoma-derived cell lines HepaRG and HepG2 and human embryonic stem cell-derived hepatocyte-like cells, are examined. For full characterization of the systems, several bioinformatics approaches are employed including gene-based, ConsensusPathDB-based and classification analysis. They provide convincingly similar outcomes, namely that upon exposure to carcinogens, the HepaRG generates a gene classifier (a gene classifier is defined as a selected set of characteristic gene signatures capable of distinguishing GTX, NGTX carcinogens and NC) able to discriminate the GTX carcinogens from the NGTX carcinogens and NC. The other in vitro models also yield cancer-relevant characteristic gene groups for the GTX exposure, but some genes are also deregulated by the NGTX carcinogens and NC. Irrespective of the tested in vitro model, the most uniformly expressed pathways following GTX exposure are the p53 and those that are subsequently induced. The NGTX carcinogens triggered no characteristic cancer-relevant gene profiles in all liver-based in vitro systems. In conclusion, liver-based in vitro models coupled with transcriptomics techniques, especially in the case when the HepaRG cell line is used, represent valuable tools for obtaining insight into the mechanism of action and identification of GTX carcinogens.
Asunto(s)
Carcinógenos/toxicidad , Hepatocitos/efectos de los fármacos , Hígado/efectos de los fármacos , Mutágenos/toxicidad , Transcriptoma/efectos de los fármacos , Animales , Carcinógenos/farmacología , Línea Celular Tumoral , Células Madre Embrionarias/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica , Células Hep G2 , Humanos , Neoplasias Hepáticas , Mutágenos/farmacología , Ratas , Ratas Sprague-Dawley , Proteína p53 Supresora de Tumor/efectos de los fármacosRESUMEN
Drug-induced liver injury induced by already approved substances is a major threat to human patients, potentially resulting in drug withdrawal and substantial loss of financial resources in the pharmaceutical industry. Trovafloxacin, a broad-spectrum fluoroquinolone, was found to have unexpected side effects of severe hepatotoxicity, which was not detected by preclinical testing. To address the limitations of current drug testing strategies mainly involving 2D cell cultures and animal testing, a three-dimensional microphysiological model of the human liver containing expandable human liver sinusoidal endothelial cells, monocyte-derived macrophages and differentiated HepaRG cells was utilized to investigate the toxicity of trovafloxacin and compared it to the structurally-related non-toxic drug levofloxacin. In the model, trovafloxacin elicited vascular and hepatocellular toxicity associated with pro-inflammatory cytokine release already at clinically relevant concentrations, whereas levofloxacin did not provoke tissue injury. Similar to in vivo, cytokine secretion was dependent on a multicellular immune response, highlighting the potential of the complex microphysiological liver model for reliably detecting drug-related cytotoxicity in preclinical testing. Moreover, hepatic glutathione depletion and mitochondrial ROS formation were elucidated as intrinsic toxicity mechanisms contributing to trovafloxacin toxicity.
Asunto(s)
Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Hepatitis , Animales , Humanos , Levofloxacino/toxicidad , Células Endoteliales , Fluoroquinolonas/toxicidad , CitocinasRESUMEN
HepaRG cells have the ability to differentiate into hepatocyte-like cells. Many papers have shown that these hepatocyte-like cells share several functional properties with intact human hepatocytes. However, although previous studies have indicated the partial maintenance of mRNA expression of drug transporters, their expression and function have not been quantitatively characterized. In the present study, the mRNA and protein expression levels and transport activities of hepatic uptake transporters, organic anion transporting polypeptides (OATPs) and Na(+)-taurocholate cotransporting polypeptide (NTCP) in HepaRG cells were compared with those in cryopreserved human hepatocytes. The mRNA expression levels of OATP1B1, OATP1B3, OATP2B1, and NTCP in HepaRG cells were 22-38%, 2-15%, 82-113%, and 191-247% of those in human hepatocytes, respectively. The relative protein expression of these transporters was comparable with their mRNA expression. We observed saturable uptake of typical substrates of NTCP and OATPs except for cholecystokinin octapeptide (OATP1B3-selective substrate), and Na(+)-dependent uptake of taurocholate was confirmed. Their relative uptake clearances were well explained by their mRNA and protein expression levels. Additionally, inhibition potencies of 12 OATP1B1 inhibitors were investigated both in HepaRG cells and in OATP1B1-expressing HEK293 cells to demonstrate the usefulness of HepaRG cells for the characterization of OATP1B1-mediated drug-drug interactions. The Ki values in both cell lines were comparable and showed significant correlation. These results suggest that the hepatic uptake transport function of OATP and NTCP transporters was relatively well maintained in HepaRG, although OATP1B3 function was too low to be detected.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Hepatocitos/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Transportadores de Anión Orgánico/metabolismo , Simportadores/metabolismo , Transporte Biológico/efectos de los fármacos , Western Blotting , Células Cultivadas , Detergentes/farmacología , Interacciones Farmacológicas , Citometría de Flujo , Células HEK293 , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Humanos , Transportador 1 de Anión Orgánico Específico del Hígado , Transportadores de Anión Orgánico/genética , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Transportadores de Anión Orgánico Sodio-Independiente/genética , Miembro 1B3 de la Familia de los Transportadores de Solutos de Aniones Orgánicos , Simportadores/genética , Ácido Taurocólico/farmacologíaRESUMEN
HepaRG cells possess the unique property to differentiate in vitro and to express various functions of mature hepatocytes, including the major cytochromes P450 (P450s). In the present study, we carefully analyzed mRNA expression and activity of the major P450s and their responsiveness to three prototypical inducers, phenobarbital, rifampicin, and omeprazole, in differentiated HepaRG cell cultures over a 4-week period after low and high seeding. Only minor differences were observed in P450 activities when measured by two cocktails of probe substrates, probably related to the choice and/or concentration of substrates. Similar results were obtained from the two cell seeding conditions. Expression and activities of several P450s were dimethyl sulfoxide-dependent. However, basal P450 expression and activities as well as their responsiveness to the prototypical inducers were well maintained over the 4-week period, and a good correlation was observed between transcript levels and corresponding activities. Thus, CYP1A2, CYP2B6, and CYP3A4 were found to accurately respond to their respective prototypical inducers, i.e., omeprazole, phenobarbital, and rifampicin. Likewise, basal expression of several phase II enzymes, transporters, and nuclear receptors, and response to inducers were also well preserved. More genes were found to be induced in HepaRG cells than in primary human hepatocytes, and no marked variation was noticed between the different passages. Taken together, these data support the conclusion that HepaRG cells represent a promising surrogate to primary human hepatocytes for xenobiotic metabolism and toxicity studies.
Asunto(s)
Sistema Enzimático del Citocromo P-450/biosíntesis , Sistema Enzimático del Citocromo P-450/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Inducción Enzimática/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Xenobióticos/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular , Línea Celular Transformada , Células Cultivadas , Sistema Enzimático del Citocromo P-450/genética , Glucuronosiltransferasa/biosíntesis , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Glutatión Transferasa/biosíntesis , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Isoenzimas/biosíntesis , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de Transporte de Membrana/biosíntesis , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Omeprazol/farmacología , Fenobarbital/farmacología , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/biosíntesis , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Reproducibilidad de los Resultados , Rifampin/farmacología , Factores de TiempoRESUMEN
Despite decades of investigation on the proliferation of adult human primary hepatocytes, their expansion in vitro still remains challenging. To later be able to consider hepatocytes as a cell therapy alternative or bridge to liver transplantation, dramatically impeded by a shortage in liver donors, the first step is having an almost unlimited source of these cells. The banking of transplantable hepatocytes also implies a protocol for their expansion that can be compatible with large-scale production. We show that adult human primary hepatocytes when grown in 3D organoids are easily amplified, providing a substantial source of functional hepatocytes ready for transplantation. Following their plating, differentiated human hepatocytes are amplified during a transient and reversible step as liver progenitors, and can subsequently be converted back to mature differentiated hepatocytes. The protocol we propose is not only compatible with automated and high-throughput cell culture systems, thanks to the expansion of hepatocytes in suspension, but also guarantees the generation of a high number of functional cells from the same patient sample, with a relatively easy set up.
Asunto(s)
Hepatocitos/citología , Organoides/citología , Células Madre/citología , Adulto , Anciano , Diferenciación Celular , Células Cultivadas , Colágeno , Combinación de Medicamentos , Femenino , Humanos , Laminina , Masculino , Proteoglicanos , Ingeniería de TejidosRESUMEN
Nanomedicines represent a promising approach in the treatment and diagnosis of numerous disorders. The majority of the injected dose of nanoparticles (NPs) is sequestrated in the liver. Despite this hepatic tropism, the interaction of NPs with the detoxification function of the liver remains unclear. The present study consists of evaluating the impact of biodegradable poly(lactide-co-glycolide) (PLGA) and silica NPs on cytochrome P450 (CYP) activities. The effects of NPs were evaluated in vitro on human and rat hepatocytes in primary cultures and in vivo by intravenous injections in healthy rats. More than the physicochemical properties, the composition of NPs (organic, inorganic) dramatically influenced the detoxification function of the liver. Silica NPs modulated the CYP activity both in rat and human hepatocytes, in contrast to PLGA NPs. A CYP isoform-dependent effect was reported and the modulation of the metabolic hepatic activity was species-dependent. Human hepatocytes were sensitive to an exposure to PLGA NPs, whereas no marked effect was detected in rat hepatocytes. The in vitro data obtained in rat hepatocytes were correlated with the in vivo data. This study emphasizes the interest to set up relevant in vitro models using human hepatic cells to evaluate the hepatotoxicity of nanomedicines.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Hepatocitos/enzimología , Nanopartículas , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Dióxido de Silicio , Animales , Hepatocitos/efectos de los fármacos , Humanos , Ácido Láctico , Cultivo Primario de Células , Ratas , Especificidad de la EspecieRESUMEN
Intrahepatic cholestasis represents a frequent manifestation of drug-induced liver injury; however, the mechanisms underlying such injuries are poorly understood. In this study of human HepaRG and primary hepatocytes, we found that bile canaliculi (BC) underwent spontaneous contractions, which are essential for bile acid (BA) efflux and require alternations in myosin light chain (MLC2) phosphorylation/dephosphorylation. Short exposure to 6 cholestatic compounds revealed that BC constriction and dilation were associated with disruptions in the ROCK/MLCK/myosin pathway. At the studied concentrations, cyclosporine A and chlorpromazine induced early ROCK activity, resulting in permanent MLC2 phosphorylation and BC constriction. However, fasudil reduced ROCK activity and caused rapid, substantial and permanent MLC2 dephosphorylation, leading to BC dilation. The remaining compounds (1-naphthyl isothiocyanate, deoxycholic acid and bosentan) caused BC dilation without modulating ROCK activity, although they were associated with a steady decrease in MLC2 phosphorylation via MLCK. These changes were associated with a common loss of BC contractions and failure of BA clearance. These results provide the first demonstration that cholestatic drugs alter BC dynamics by targeting the ROCK/MLCK pathway; in addition, they highlight new insights into the mechanisms underlying bile flow failure and can be used to identify new predictive biomarkers of drug-induced cholestasis.
Asunto(s)
Canalículos Biliares/efectos de los fármacos , Miosinas Cardíacas/metabolismo , Clorpromazina/farmacología , Ciclosporina/farmacología , Cadenas Ligeras de Miosina/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , Quinasas Asociadas a rho/metabolismo , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Ácidos y Sales Biliares/metabolismo , Canalículos Biliares/fisiología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Microscopía Fluorescente , Miosina Tipo II/metabolismo , Fosforilación/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Imagen de Lapso de Tiempo , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
The role of hepatobiliary transporters in drug-induced liver injury remains poorly understood. Various in vivo and in vitro biological approaches are currently used for studying hepatic transporters; however, appropriate localization and functional activity of these transporters are essential for normal biliary flow and drug transport. Human hepatocytes (HHs) are considered as the most suitable in vitro cell model but erratic availability and inter-donor functional variations limit their use. In this work, we aimed to compare localization of influx and efflux transporters and their functional activity in differentiated human HepaRG hepatocytes with fresh HHs in conventional (CCHH) and sandwich (SCHH) cultures. All tested influx and efflux transporters were correctly localized to canalicular [bile salt export pump (BSEP), multidrug resistance-associated protein 2 (MRP2), multidrug resistance protein 1 (MDR1), and MDR3] or basolateral [Na(+)-taurocholate co-transporting polypeptide (NTCP) and MRP3] membrane domains and were functional in all models. Contrary to other transporters, NTCP and BSEP were less abundant and active in HepaRG cells, cellular uptake of taurocholate was 2.2- and 1.4-fold and bile excretion index 2.8- and 2.6-fold lower, than in SCHHs and CCHHs, respectively. However, when taurocholate canalicular efflux was evaluated in standard and divalent cation-free conditions in buffers or cell lysates, the difference between the three models did not exceed 9.3%. Interestingly, cell imaging showed higher bile canaliculi contraction/relaxation activity in HepaRG hepatocytes and larger bile canaliculi networks in SCHHs. Altogether, our results bring new insights in mechanisms involved in bile acids accumulation and excretion in HHs and suggest that HepaRG cells represent a suitable model for studying hepatobiliary transporters and drug-induced cholestasis.
Asunto(s)
Hepatocitos/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Línea Celular , HumanosRESUMEN
The EU FP6 project carcinoGENOMICS explored the combination of toxicogenomics and in vitro cell culture models for identifying organotypical genotoxic- and non-genotoxic carcinogen-specific gene signatures. Here the performance of its gene classifier, derived from exposure of metabolically competent human HepaRG cells to prototypical non-carcinogens (10 compounds) and hepatocarcinogens (20 compounds), is reported. Analysis of the data at the gene and the pathway level by using independent biostatistical approaches showed a distinct separation of genotoxic from non-genotoxic hepatocarcinogens and non-carcinogens (up to 88 % correct prediction). The most characteristic pathway responding to genotoxic exposure was DNA damage. Interlaboratory reproducibility was assessed by blindly testing of three compounds, from the set of 30 compounds, by three independent laboratories. Subsequent classification of these compounds resulted in correct prediction of the genotoxicants. As expected, results on the non-genotoxic carcinogens and the non-carcinogens were less predictive. In conclusion, the combination of transcriptomics with the HepaRG in vitro cell model provides a potential weight of evidence approach for the evaluation of the genotoxic potential of chemical substances.
RESUMEN
The HepaRG cell line is the first human cell line able to differentiate in vitro into mature hepatocyte-like cells. Our main objective within the framework of the EEC-LIINTOP project was to optimize the use of this cell line for drug metabolism and toxicity studies, especially after repeat treatments. The main results showed that differentiated HepaRG cells: (i) retained their drug metabolism capacity (major CYPs, phase 2 enzymes, transporters and nuclear receptors) and responsiveness to prototypical inducers at relatively stable levels for several weeks at confluence. The levels of several functions, including some CYPs such as CYP3A4, were dependent on the addition of dimethyl sulfoxide in the culture medium; (ii) sustained the different types of chemical-induced hepatotoxicity, including steatosis, phospholipidosis and cholestasis, after acute and/or repeat treatment with reference drugs. In particular, drug-induced vesicular steatosis was demonstrated in vitro for the first time. In conclusion, our results from the LIINTOP project, together with other studies reported concomitantly or more recently in the literature, support the conclusion that the metabolically competent human HepaRG cells represent a surrogate to primary human hepatocytes for investigating drug metabolism parameters and both acute and chronic effects of xenobiotics in human liver.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Hepatocitos/metabolismo , Modelos Biológicos , Diferenciación Celular , Línea Celular , Enfermedad Hepática Inducida por Sustancias y Drogas/fisiopatología , Sistema Enzimático del Citocromo P-450/metabolismo , Dimetilsulfóxido/química , Hepatocitos/enzimología , Humanos , Factores de Tiempo , Xenobióticos/administración & dosificación , Xenobióticos/metabolismo , Xenobióticos/toxicidadRESUMEN
Microinjection is the most flexible transfection method in terms of choice of reagents to inject into cells. But this method lacks the high throughput to compete with less flexible methods like chemical- or viral-based approaches. Various approaches have been pursued to increase the throughput by automating the microinjection process. However, these approaches focused solely on the microinjection itself and disregarded the tasks before and after the injection, which also belong to the critical time path of the whole process, that is, sorting out viable cells from a cell suspension, placing the cell for injection, and collecting the cell after the injection. In the approach with our XenoFactor, we demonstrate a system capable of running the whole process automatically. By optimizing the XenoFactor for Xenopus laevis oocytes, we could demonstrate the successful automated injection. Starting from a suspension with a mixture of defolliculated oocytes at different stages and quality levels, the manual approach requires 1 day in total for the preparation of 400 microinjected oocytes. The XenoFactor takes only 4h for the same amount and delivers injected oocytes of reproducible quality and without the fatigue symptoms experienced during the manual approach.