Cytometric analysis for drug-induced steatosis in HepG2 cells |
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Authors: | M Teresa Donato Alicia Martínez-Romero Alejandro Negro José V Castell José-Enrique O’Connor |
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Institution: | a Unidad de Hepatología Experimental, Centro de Investigación, Hospital La Fe, Avda Campanar 21, 46009 Valencia, Spain b CIBEREHD, FIS, Spain c Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Valencia, Avda Blasco Ibáñez 15, 46010 Valencia, Spain d Laboratorio de Citómica, Unidad Mixta CIPF-UVEG, Centro de Investigación Príncipe Felipe, Avda. Autopista del Saler 16, 46013 Valencia, Spain |
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Abstract: | Drugs are capable of inducing hepatic lipid accumulation. When fat accumulates, lipids are primarily stored as triglycerides which results in steatosis and provides substrates for lipid peroxidation. An in vitro multiparametric flow cytometry assay was performed in HepG2 cells by using fluorescent probes to analyze cell viability (propidium iodide, PI), lipid accumulation (BODIPY493/503), mitochondrial membrane potential (tetramethyl rhodamine methyl ester, TMRM) and reactive oxygen species generation (ROS) (2′,7′-dihydrochlorofluorescein diacetate, DHCF-DA) as functional markers. All the measurements were restricted to live cells by gating the cells that excluded PI or those that exhibited the typical forward and side scatter features of live cells. The assay was qualified by analyzing a number of selected model drugs with a well documented induction of steatosis in vivo using different mechanisms as positive controls and several non-steatosic compounds as negative controls. For the cytometric screening assay, the concentrations tested were up to the corresponding IC10 value determined by the MTT assay. Among the parameters analyzed, increased BODIPY fluorescence was the most sensitive and selective marker of drug-induced steatosis. However, a more consistent predictive approach was the combination of two endpoints: lipid accumulation and ROS generation. The assay correctly identified 100% of steatosis-positive and steatosis-negative compounds, and a high steatosis risk was predicted for amiodarone, doxycycline, tetracycline and valproate treatments at therapeutic doses. The results suggest that this cell-based assay may be a useful approach to identify the potential of drug candidates to induce steatosis. |
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Keywords: | Drug-induced steatosis HepG2 cells Multiparametric assay Flow cytometry In vitro hepatotoxicity |
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