title: Automated detection of hepatotoxic compounds in human hepatocytes using HepaRG cells and image-based analysis of mitochondrial dysfunction with JC-1 dye.
abstract: 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.
authors.affiliation: Inserm UMR 991, Université de Rennes 1, Faculté de médecine, F-35043 Rennes Cedex, France. keligpernelle@hotmail.fr
authors.forename: K
authors.initials: K
authors.lastname: Pernelle
authors.forename: R
authors.initials: R
authors.lastname: Le Guevel
authors.forename: D
authors.initials: D
authors.lastname: Glaise
authors.forename: C Gaucher-Di
authors.initials: CG
authors.lastname: Stasio
authors.forename: T
authors.initials: T
authors.lastname: Le Charpentier
authors.forename: B
authors.initials: B
authors.lastname: Bouaita
authors.forename: A
authors.initials: A
authors.lastname: Corlu
authors.forename: C
authors.initials: C
authors.lastname: Guguen-Guillouzo
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000032:analysis / Q000037:antagonists & inhibitors; D001562:Benzimidazoles / Q000032:analysis*; D002232:Carbocyanines / Q000032:analysis*; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D018920:Coculture Techniques; D064420:Drug-Related Side Effects and Adverse Reactions / Q000175:diagnosis / Q000378:metabolism; D005456:Fluorescent Dyes / Q000032:analysis*; D022781:Hepatocytes / Q000737:chemistry* / Q000187:drug effects; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods* / Q000592:standards; D008856:Microscopy, Fluorescence / Q000379:methods; D008928:Mitochondria / Q000737:chemistry* / Q000187:drug effects; D018675:Toxicity Tests / Q000379:methods* / Q000592:standards; D014700:Verapamil / Q000032:analysis / Q000494:pharmacology
chemical_list: D018435:ATP Binding Cassette Transporter, Subfamily B; D001562:Benzimidazoles; D002232:Carbocyanines; D005456:Fluorescent Dyes; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; D014700:Verapamil