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In vitro assay for drug‐induced hepatosteatosis using rat primary hepatocytes, a fluorescent lipid analog and gene expression analysis
Author(s) -
Fujimura Hisako,
Murakami Naoko,
Kurabe Michie,
Toriumi Wataru
Publication year - 2009
Publication title -
journal of applied toxicology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.784
H-Index - 87
eISSN - 1099-1263
pISSN - 0260-437X
DOI - 10.1002/jat.1420
Subject(s) - clofibrate , chemistry , lipid metabolism , fluorescence , hepatocyte , peroxisome , lipid droplet , gene expression , in vitro , fluorescence microscope , biochemistry , drug metabolism , viability assay , pharmacology , microbiology and biotechnology , receptor , metabolism , biology , gene , physics , quantum mechanics
Abstract To evaluate new drugs' potential for hepatosteatosis, we developed a cell‐based assay using a fluorescent fatty acid analog: BODIPY558/568 C 12 . Rat primary hepatocytes were exposed to positive reference compounds [cyclosporine A (CsA), clofibrate (CFR), tetracycline (TC), valproic acid (VPA), carbon tetrachloride (CCl4), tamoxifen (TMX)] in the presence of BODIPY558/568 C 12 . The formation of fluorsecent particles or lipid droplets in the cytoplasm was confirmed by confocal laser scanning microscopy and electron microscopy respectively. The accumulation of BODIPY558/568 C 12 was measured by fluorometry and high content imaging method. All positive reference compounds increased fluorescent particles in number and fluorescence intensity. High content imaging was more sensitive and selective method than fluorometry to detect fluorescent particles. Gene expression analysis of the hepatocytes showed two patterns: genes related to lipid metabolism/synthesis were down‐regulated by oxidative stress inducing compounds: CsA, TC and TMX, and up‐regulated by peroxisome proliferator‐activated receptor‐alpha agonists: CFR and VPA. From these findings, we concluded that the cell‐based assay developed in this study is an appropriate method to predict drugs' potential for hepatosteatosis, and gene expression analysis is useful to profile the mechanism of the hepatosteatosis. Copyright © 2009 John Wiley & Sons, Ltd.