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

Journal Article · Mon Aug 01 00:00:00 EDT 2011 · Toxicology and Applied Pharmacology

DOI:https://doi.org/10.1016/j.taap.2011.04.018· OSTI ID:21587796

Pernelle, K ^[1]; Glaise, D ^[1]; Bouaita, B ^[2]; Corlu, A ^[1]; Guguen-Guillouzo, C ^[1]

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1. Inserm UMR 991, Universite de Rennes 1, Faculte de medecine, F-35043 Rennes Cedex (France)
2. Biopredic international, Parc d'activite Breteche batA4, 35760 Saint-Gregoire (France)

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. - Highlights: > We define conditions to preserve differentiation of selective pure HepaRG hepatocyte cultures. > In these conditions, CYPs, transporters expression and activity were similar to a mixed HepaRG population. > Long term differentiation period before HepaRG hepatocyte isolation influence this establishment. > These conditions were settled to develop a hepatoxicity test using automated-imaging analysis. > JC-1 detects MPT, an early event of cell death, in altered HepaRG hepatocytes when efflux of this dye by MDR1 is inhibited.

• OSTI ID: 21587796
• Journal Information: Toxicology and Applied Pharmacology, Vol. 254, Issue 3; Other Information: DOI: 10.1016/j.taap.2011.04.018; PII: S0041-008X(11)00167-0; Copyright (c) 2011 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA); ISSN 0041-008X
• Country of Publication: United States
• Language: English

Related Subjects

60 APPLIED LIFE SCIENCES
APOPTOSIS
CELL DIFFERENTIATION
DETECTION
DRUGS
DYES
ENZYMES
FLUORESCENCE
HUMAN POPULATIONS
IMAGES
IMPURITIES
LIVER
LIVER CELLS
MITOCHONDRIA
NECROSIS
TOXICITY
ANIMAL CELLS
BODY
CELL CONSTITUENTS
DIGESTIVE SYSTEM
EMISSION
GLANDS
LUMINESCENCE
ORGANIC COMPOUNDS
ORGANS
PATHOLOGICAL CHANGES
PHOTON EMISSION
POPULATIONS
PROTEINS
SOMATIC CELLS