RESUMO
Drug-induced liver injury is a major cause of drug discovery failure in clinical trials and a leading cause of liver disease. Current preclinical drug testing does not predict hepatotoxicity which highlights the importance of developing highly predictive cell-based models. The use of stem cell technology and differentiation into hepatocyte-like cells (HLCs) could provide a stable source of hepatocytes for multiple applications, including drug screening. HLCs derived from both embryonic and induced pluripotent stem cells have been used to accurately predict hepatotoxicity as well as to test individual-specific toxicity. Although there are still many limitations, mainly related to the lack of fully maturity of the HLCs derived from pluripotent stem cells, they could provide a relative unlimited and consistent supply of cells with stable phenotype, that could be obtained from different donors, enabling the generation of a library of HLCs representative of the variability of human population.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/diagnóstico , Avaliação Pré-Clínica de Medicamentos/métodos , Células-Tronco Embrionárias/citologia , Hepatócitos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Diferenciação Celular , Células-Tronco Embrionárias/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , FenótipoRESUMO
Drug-induced liver injury (DILI) has a considerable impact on human health and is a major challenge in drug safety assessments. DILI is a frequent cause of liver injury and a leading reason for post-approval drug regulatory actions. Considerable variations in the expression levels of both cytochrome P450 (CYP) and conjugating enzymes have been described in humans, which could be responsible for increased susceptibility to DILI in some individuals. We herein explored the feasibility of the combined use of HepG2 cells co-transduced with multiple adenoviruses that encode drug-metabolising enzymes, and a high-content screening assay to evaluate metabolism-dependent drug toxicity and to identify metabolic phenotypes with increased susceptibility to DILI. To this end, HepG2 cells with different expression levels of specific drug-metabolism enzymes (CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, GSTM1 and UGT2B7) were exposed to nine drugs with reported hepatotoxicity. A panel of pre-lethal mechanistic parameters (mitochondrial superoxide production, mitochondrial membrane potential, ROS production, intracellular calcium concentration, apoptotic nuclei) was used. Significant differences were observed according to the level of expression and/or the combination of several drug-metabolism enzymes in the cells created ad hoc according to the enzymes implicated in drug toxicity. Additionally, the main mechanisms implicated in the toxicity of the compounds were also determined showing also differences between the different types of cells employed. This screening tool allowed to mimic the variability in drug metabolism in the population and showed a highly efficient system for predicting human DILI, identifying the metabolic phenotypes associated with increased DILI risk, and indicating the mechanisms implicated in their toxicity.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/etiologia , Família 2 do Citocromo P450/genética , Avaliação Pré-Clínica de Medicamentos/métodos , Testes de Toxicidade/métodos , Adenoviridae/genética , Família 2 do Citocromo P450/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Ensaios de Triagem em Larga Escala/métodos , Humanos , Inativação Metabólica/genética , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
La hepatotoxicidad idiosincrática se presenta de forma impredecible en un grupo reducido de individuos susceptibles y sin relación directa con la dosis. Si bien los mecanismos implicados en estas reacciones adversas de los fármacos permanecen sin esclarecer, se han propuesto varias hipótesis para explicar su naturaleza idiosincrática, entre las que se incluyen la conversión del fármaco en metabolitos reactivos (bioactivación), la activación del sistema inmune, la disfunción mitocondrial o el estrés inflamatorio. Los estudios de seguridad que se realizan durante el desarrollo de fármacos son insuficientes para predecir el daño tóxico de naturaleza idiosincrática. Los actuales modelos preclínicos (animales de experimentación e in vitro) son incapaces de reproducir los múltiples factores genéticos o adquiridos que influyen sobre la susceptibilidad individual, lo que hace prácticamente imposible la detección de fármacos con capacidad de inducir episodios idiosincráticos. En los últimos años se viene realizado un gran esfuerzo dirigido a desarrollar nuevas estrategias capaces de anticipar el daño hepático idiosincrático inducido por fármacos e identificar aquellos pacientes que presentan mayor riesgo a la hepatotoxicidad. Los ensayos in vitro basados en el uso de modelos celulares que reflejen la idiosincrasia individual, junto con las tecnologías ómicas y de análisis celular multiparamétrico, han reorientado la forma de evaluar los efectos tóxicos de los fármacos y se postulan como herramientas con gran proyección para discriminar el potencial hepatotóxico de nuevos fármacos (AU)
Idiosyncratic hepatotoxicity occurs unpredictably in a small group of susceptible individuals with no direct dose relationship. Although the mechanisms involved in these adverse drug reactions remain unclear, several hypotheses have been proposed to explain their idiosyncratic nature, including drug conversion into reactive metabolites (bioactivation), activation of the immune system, mitochondrial dysfunction or inflammatory stress. Safety studies conducted during drug development are clearly insufficient to predict idiosyncratic toxic damage. The current preclinical models (experimental animals and in vitro) are unable to reproduce the multiple genetic or acquired factors that influence the individual susceptibility, which makes it practically impossible to detect drugs with the capacity to induce idiosyncratic episodes. In recent years, a great effort has been made to develop new strategies capable of anticipating idiosyncratic liver damage induced by drugs and to identify those patients with an increased risk for hepatotoxicity. In vitro assays based on the use of cellular models that reflect individual idiosyncrasy, together with omics and multiparametric cellular analysis technologies, have reoriented the evaluation of the toxic effects induced by drugs and are postulated as tools with great projection to discriminate hepatotoxic potential of new drugs (AU)
Assuntos
Humanos , Idiossincrasia , Hepatócitos , Preparações Farmacêuticas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/fisiopatologia , Técnicas In Vitro/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Drogas em Investigação , Disponibilidade BiológicaRESUMO
Disruption of the vectorial bile acid transport in the liver is a key feature of cholestatic drugs, although many causal and mechanistic aspects are still unknown. The aim of the present study was to explore if cholestatic drugs can repress or induce the expression of hepatic transporters. To this end, sandwich-cultured rat hepatocytes were treated with cholestatic and non-cholestatic (steatotic, non-hepatotoxic, etc.) drugs and the mRNA expression of 10 uptake and efflux biliary transporters was measured. Results evidenced that all cholestatic drugs cause extensive alterations in the mRNA expression of most biliary transporters. Surprisingly, nearly all steatotic drugs also affected the expression of these genes. The most frequent alterations triggered by both types of drugs were the repression of OATP1A1, NTCP and BSEP, and the induction of MRP2/3/4, MDR2 and ABCG5/8. The majority of these alterations were also observed in vivo, in the livers of treated rats. The common signature of cholestatic and steatotic drugs was the repression of OATP1A1. Indeed, ROC curve analysis indicated that OATP1A1 mRNA is a very sensitive marker to identify drugs with cholestatic or steatotic potential, with a maximal sensitivity and specificity of 0.917 and 0.941, respectively. We conclude that alteration of expression of hepatobiliary transporters is a hallmark of both cholestatic and steatotic drugs, lending support to a connection between these two mechanisms of hepatotoxicity. Moreover, OATP1A1 mRNA is proposed as a very simple and useful screening biomarker for the prediction of new cholestatic or steatotic drugs in early drug development.
Assuntos
Bile/metabolismo , Biomarcadores/análise , Proteínas de Transporte/metabolismo , Colestase/induzido quimicamente , Avaliação Pré-Clínica de Medicamentos/métodos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Fígado Gorduroso/induzido quimicamente , Hepatócitos/efeitos dos fármacos , RNA Mensageiro/biossíntese , Animais , Bile/efeitos dos fármacos , Proteínas de Transporte/biossíntese , Células Cultivadas , Masculino , Transportadores de Ânions Orgânicos Sódio-Independentes/análise , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Valor Preditivo dos Testes , Ratos , Ratos Sprague-Dawley , Tetraciclinas/toxicidadeRESUMO
Only a few in vitro assays have been proposed to evaluate the steatotic potential of new drugs. The present study examines the utility of HepaRG cells as a cell-based assay system for screening drug-induced liver steatosis. A high-content screening assay was run to evaluate multiple toxicity-related cell parameters in HepaRG cells exposed to 28 compounds, including drugs reported to cause steatosis through different mechanisms and non-steatotic compounds. Lipid content was the most sensitive parameter for all the steatotic drugs, whereas no effects on lipid levels were produced by non-steatotic compounds. Apart from fat accumulation, increased ROS production and altered mitochondrial membrane potential were also found in the cells exposed to steatotic drugs, which indicates that all these cellular events contributed to drug-induced hepatotoxicity. These findings are of clinical relevance as most effects were observed at drug concentrations under 100-fold of the therapeutic peak plasmatic concentration. HepaRG cells showed increased lipid overaccumulation vs. HepG2 cells, which suggests greater sensitivity to drug-induced steatosis. An altered expression profile of transcription factors and the genes that code key proteins in lipid metabolism was also found in the cells exposed to drugs capable of inducing liver steatosis. Our results generally indicate the value of HepaRG cells for assessing the risk of liver damage associated with steatogenic compounds and for investigating the molecular mechanisms involved in drug-induced steatosis.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Fígado Gorduroso/induzido quimicamente , Linhagem Celular Tumoral , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Humanos , Metabolismo dos Lipídeos/genéticaRESUMO
Drug-induced liver injury (DILI) is a significant leading cause of hepatic dysfunction, drug failure during clinical trials and post-market withdrawal of approved drugs. Many cases of DILI are unexpected reactions of an idiosyncratic nature that occur in a small group of susceptible individuals. Intensive research efforts have been made to understand better the idiosyncratic DILI and to identify potential risk factors. Metabolic bioactivation of drugs to form reactive metabolites is considered an initiation mechanism for idiosyncratic DILI. Reactive species may interact irreversibly with cell macromolecules (covalent binding, oxidative damage), and alter their structure and activity. This review focuses on proposed in vitro screening strategies to predict and reduce idiosyncratic hepatotoxicity associated with drug bioactivation. Compound incubation with metabolically competent biological systems (liver-derived cells, subcellular fractions), in combination with methods to reveal the formation of reactive intermediates (e.g., formation of adducts with liver proteins, metabolite trapping or enzyme inhibition assays), are approaches commonly used to screen the reactivity of new molecules in early drug development. Several cell-based assays have also been proposed for the safety risk assessment of bioactivable compounds. Copyright © 2015 John Wiley & Sons, Ltd.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Avaliação Pré-Clínica de Medicamentos , Drogas em Investigação/efeitos adversos , Fígado/efeitos dos fármacos , Microssomos Hepáticos/efeitos dos fármacos , Modelos Biológicos , Ativação Metabólica , Animais , Técnicas de Cultura de Células/tendências , Linhagem Celular , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/epidemiologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Técnicas de Cocultura/tendências , Avaliação Pré-Clínica de Medicamentos/tendências , Drogas em Investigação/química , Drogas em Investigação/farmacocinética , Humanos , Técnicas In Vitro/tendências , Fígado/citologia , Fígado/metabolismo , Fígado/patologia , Microfluídica/métodos , Microfluídica/tendências , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/patologia , Proteínas Recombinantes/metabolismo , Medição de Risco , Fatores de Risco , Alicerces Teciduais/tendênciasRESUMO
OBJECTIVE: Mango (Mangifera indica L.) stem bark extract (MSBE) is a natural product with biological properties and mangiferin is the major component. This paper reported the evaluation of the protective effects of MSBE and mangiferin against the toxicity induced in HepG2 cells by tert-butyl hydroperoxide or amiodarone. METHOD: Nuclear morphology, cell viability, intracellular calcium concentration and reactive oxygen species (ROS) production were measured by using a high-content screening multiparametric assay. KEY FINDINGS: MSBE and mangiferin produced no toxicity below 500 mg/ml doses. A marked recovery in cell viability, which was reduced by the toxicants, was observed in cells pre-exposed to MSBE or mangiferin at 5-100 mg/ml doses. We also explored the possible interaction of both products over P-glycoprotein (P-gp). MSBE and mangiferin above 100 mg/ml inhibited the activity of P-gp in HepG2 cells. CONCLUSIONS: MSBE and mangiferin showed cytoprotective effects of against oxidative damage and mitochondrial toxicity induced by xenobiotics to human hepatic cells but it seemed that other constituents of the extract could contribute to MSBE protective properties. In addition, the drug efflux should be taken into account because of the inhibition of the P-gp function observed in those cells exposed to both natural products.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Extratos Vegetais/farmacologia , Xantonas/farmacologia , Amiodarona/toxicidade , Cálcio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Hep G2 , Humanos , Mangifera , Casca de Planta , Extratos Vegetais/administração & dosagem , Extratos Vegetais/toxicidade , Caules de Planta , Espécies Reativas de Oxigênio/metabolismo , Xantonas/administração & dosagem , Xantonas/isolamento & purificação , terc-Butil Hidroperóxido/toxicidadeRESUMO
Fluorimetric methods to assess cytochrome P450 (P450) activities that do not require metabolite separation have been developed. These methods make use of non- or low-fluorescent P450 substrates that produce highly fluorescent metabolites in aqueous solutions. The assays are based on the direct incubation of intact cells in culture with appropriate fluorogenic probe substrates, followed by fluorimetric quantification of the product formed and released into incubation medium. We describe a battery of fluorescence assays for rapid measurement of the activity of nine P450s involved in drug metabolism. For each individual P450 activity the probe showing the best properties (highest metabolic rates, lowest background fluorescence) has been selected. Fluorescence-based assays are highly sensitive and allow the simultaneous activity assessments of cells cultured in 96-well plates, using plate readers, with notable reductions in costs, time, and cells, thus enhancing sample throughput.
Assuntos
Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios Enzimáticos/métodos , Fluorometria/métodos , Calibragem , Técnicas de Cultura de Células , Linhagem Celular , Sistema Enzimático do Citocromo P-450/biossíntese , Indução Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Hepatócitos/enzimologia , HumanosRESUMO
The liver plays a key role in fat metabolism, and excessive lipid accumulation in liver cells is characterised by a large spectrum of lesions, e.g., steatosis and phospholipidosis. Steatosis is increased lipid accumulation, mainly as triglycerides, in the liver, while phospholipidosis is a lysosomal storage disorder characterised by intracellular accumulation of phospholipids. These alterations can be induced by several factors, including exposure to certain drugs. Drug-induced steatosis is often reversible, and prolonged exposure to certain drugs can cause macrovacuolar steatosis, a benign hepatic lesion, that can evolve into steatohepatitis and cirrhosis in some patients. Some drugs may acutely induce microvesicular steatosis which, despite having a good short-term prognosis, can lead to chronic lipid peroxidation and to the development of steatohepatitis lesions with time. Over 50 marketed drugs have been reported to induce phospholipidosis in different tissues, including the liver. Although drug-induced phospholipidosis is often reversible and there is no definitive evidence for its toxicological implications, it is considered an adverse side finding by regulatory agencies. As developing new drugs is a complex, lengthy and expensive process that aims to identify pharmacologically active, low-toxicity drug candidates among closely related compounds, it could be advantageous to determine which drugs are able to induce lipid metabolic disorders in early developmental stages. To this end, in vitro predictive screening assays, particularly cell-based approaches in which many drug candidates are evaluated, have been developed to identify and rule out compounds with a strong liver steatosis and/or phospholipidosis-inducing potential.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Fígado Gorduroso/induzido quimicamente , Transtornos do Metabolismo dos Lipídeos/induzido quimicamente , Animais , Bioensaio , Fígado Gorduroso/metabolismo , Humanos , Transtornos do Metabolismo dos Lipídeos/metabolismo , Fígado/citologia , Fosfolipídeos/metabolismoRESUMO
In the present study, we developed a cell-based protocol for the identification of drugs able to induce steatosis. The assay measures multiple markers of toxicity in a 96-well plate format using high-content screening (HCS) technology. After treating HepG2 cells with increasing concentrations of the tested compounds, toxicity parameters were analyzed using fluorescent probes: BODIPY493/503 (lipid content), 2',7'-dihydrodichlorofluorescein diacetate (reactive oxygen species [ROS] generation), tetramethyl rhodamine methyl ester (mitochondrial membrane potential), propidium iodide (cell viability), and Hoechst 33342 (nuclei staining). A total of 16 drugs previously reported to induce liver steatosis through different mechanisms (positive controls) and six nonsteatotic compounds (negative controls) were included in the study. All the steatosis-positive compounds significantly increased BODIPY493/503 fluorescence in HepG2 cells, whereas none of the negative controls induced lipid accumulation. In addition to effects on fat levels, increased ROS generation was produced by certain compounds, which could be indicative of increased risk of liver damage. Our results suggest that this in vitro approach is a simple, rapid, and sensitive screening tool for steatosis-inducing drugs. This conclusion should be confirmed by testing a larger number of steatosis-positive and -negative inducers.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Fígado Gorduroso/induzido quimicamente , Microscopia de Fluorescência , Biomarcadores/análise , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Corantes Fluorescentes , Células Hep G2 , Humanos , Fígado/patologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: Storage of human hepatocytes is essential for their use in research and liver cell transplantation. However, cryopreservation and thawing (C/T) procedures have detrimental effects on the viability and functionality compared with fresh cells. The aim of this study was to upgrade the standard C/T methodology to obtain better quality hepatocytes for cell transplantation to improve the overall clinical outcome. METHODS: Human hepatocytes isolated from donor livers were cryopreserved in University of Wisconsin solution with 10% dimethyl sulfoxide (standard medium), which was supplemented with 10% or 20% of platelet lysate. Thawing media supplemented with up to 30 mM glucose was also investigated. The effects on cell viability, adhesion proteins (e-cadherin, ß-catenin, and ß1-integrin) expression, attachment efficiency, apoptotic indicators, Akt signaling, ATP levels, and cytochrome P450 activities have been evaluated. RESULTS: The results indicate that the hepatocytes cryopreserved in a medium supplemented with platelet lysate show better recovery than those preserved in the standard medium: higher expression of adhesion molecules, higher attachment efficiency and cell survival; decreased number of apoptotic nuclei and caspase-3 activation; maintenance of ATP levels; and drug biotransformation capability close to those in fresh hepatocytes. Supplementation of thawing media with glucose led to a significant decrease in caspase-3 activation and to increased adhesion molecules preservation and Akt signal transduction after C/T. Minor nonsignificant changes in cell viability and attachment efficiency were observed. CONCLUSIONS: These promising results could lead to a new cryopreservation procedure to improve human hepatocyte cryopreservation outcome.
Assuntos
Plaquetas/citologia , Criopreservação/métodos , Hepatócitos/citologia , Manejo de Espécimes/métodos , Adenosina/farmacologia , Trifosfato de Adenosina/metabolismo , Alopurinol/farmacologia , Apoptose , Caspase 3/metabolismo , Núcleo Celular/metabolismo , Sobrevivência Celular , Transplante de Células/métodos , Ativação Enzimática , Congelamento , Glutationa/farmacologia , Humanos , Insulina/farmacologia , Fígado/metabolismo , Soluções para Preservação de Órgãos/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Rafinose/farmacologia , Transdução de Sinais , Resultado do TratamentoRESUMO
Liver grafts discarded for transplantation because of macrosteatosis can constitute a valuable source of human hepatocytes for in vitro metabolic and pharmacotoxicological studies or for therapeutic applications. A condition for using hepatocyte suspensions for these purposes is the preservation of their metabolic competence and, particularly, drug-metabolizing enzymes. A reduction in microsomal cytochrome P450 (P450) activities was observed in fatty livers (>40% steatosis) with respect to normal tissue. Similarly, decreased levels of 7-ethoxycoumarin O-deethylation and testosterone metabolism were observed in human hepatocyte cultures prepared from steatotic liver tissue. To clarify the potential impact of lipid accumulation on human hepatic P450 enzymes, we have used an in vitro model of "cellular steatosis" by incubation of cultured hepatocytes with increasing concentrations (0.25-3 mM) of long-chain free fatty acids (FFA). A dose-dependent accumulation of lipids in the cytosol is induced by FFA mixture. Hepatocytes exposed to 1 mM FFA for 14 h showed lower activity values of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 enzymes than nontreated hepatocytes (about 45-65% reduction). This treatment also produced significant decreases in CYP1A2, CYP2A6, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 mRNA to about 55 to 75% of mRNA levels in control cells. Our results suggest that although human hepatocytes isolated from steatotic liver show reduced P450 activities, they are metabolically competent and can be used for drug metabolism studies.