Automated detection of hepatotoxic compounds in human hepatocytes using HepaRG cells and image-based analysis of mitochondrial dysfunction with JC-1 dye

Autor: Christiane Guguen-Guillouzo, T. Le Charpentier, K. Pernelle, B. Bouaita, R. Le Guével, Denise Glaise, Anne Corlu, C. Gaucher-Di Stasio
Přispěvatelé: Foie, métabolismes et cancer, Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), INSERM, CNRS, European Community 6th Framwork project (COMICS no. 037575), Université de Rennes (UR)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )
Jazyk: angličtina
Rok vydání: 2011
Předmět:
MESH: Microscopy
Fluorescence
Toxicology
MPT
MESH: Drug Toxicity
Imaging
MESH: Hepatocytes
0302 clinical medicine
JC-1
Image Processing
Computer-Assisted
Hepatocyte
Cells
Cultured
0303 health sciences
Confluency
Cell Differentiation
Carbocyanines
MESH: Fluorescent Dyes
MESH: Image Processing
Computer-Assisted
Mitochondria
medicine.anatomical_structure
Biochemistry
030220 oncology & carcinogenesis
Differentiation
[SDV.TOX]Life Sciences [q-bio]/Toxicology
Toxicity
Efflux
MESH: Carbocyanines
MESH: P-Glycoproteins
MESH: Cells
Cultured
MESH: Cell Differentiation
ATP Binding Cassette Transporter
Subfamily B
MESH: Cell Line
Tumor
Drug-Related Side Effects and Adverse Reactions
MESH: Mitochondria
Biology
MESH: Coculture Techniques
03 medical and health sciences
Cell Line
Tumor
Toxicity Tests
medicine
Humans
MESH: Toxicity Tests
030304 developmental biology
Fluorescent Dyes
Pharmacology
MESH: Humans
HepaRG cell
Coculture Techniques
Multiple drug resistance
Microscopy
Fluorescence
Verapamil
Cell culture
Apoptosis
Hepatic stellate cell
Hepatocytes
Benzimidazoles
MESH: Verapamil
MESH: Benzimidazoles
Zdroj: Toxicology and Applied Pharmacology
Toxicology and Applied Pharmacology, Elsevier, 2011, 254 (3), pp.256-66. ⟨10.1016/j.taap.2011.04.018⟩
Toxicology and Applied Pharmacology, 2011, 254 (3), pp.256-66. ⟨10.1016/j.taap.2011.04.018⟩
ISSN: 0041-008X
1096-0333
DOI: 10.1016/j.taap.2011.04.018⟩
Popis: International audience; In this study, our goal was to develop an efficient in situ test adapted to screen hepatotoxicity of various chemicals, a process which remains challenging during the early phase of drug development. The test was based on functional human hepatocytes using the HepaRG cell line, and automation of quantitative fluorescence microscopy coupled with automated imaging analysis. Differentiated HepaRG cells express most of the specific liver functions at levels close to those found in primary human hepatocytes, including detoxifying enzymes and drug transporters. A triparametric analysis was first used to evaluate hepatocyte purity and differentiation status, mainly detoxication capacity of cells before toxicity testing. We demonstrated that culturing HepaRG cells at high density maintained high hepatocyte purity and differentiation level. Moreover, evidence was found that isolating hepatocytes from 2-week-old confluent cultures limited variations associated with an ageing process occurring over time in confluent cells. Then, we designed a toxicity test based on detection of early mitochondrial depolarisation associated with permeability transition (MPT) pore opening, using JC-1 as a metachromatic fluorescent dye. Maximal dye dimerization that would have been strongly hampered by efficient efflux due to the active, multidrug-resistant (MDR) pump was overcome by coupling JC-1 with the MDR inhibitor verapamil. Specificity of this test was demonstrated and its usefulness appeared directly dependent on conditions supporting hepatic cell competence. This new hepatotoxicity test adapted to automated, image-based detection should be useful to evaluate the early MPT event common to cell apoptosis and necrosis and simultaneously to detect involvement of the multidrug resistant pump with target drugs in a human hepatocyte environment.
Databáze: OpenAIRE
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