In vitro assay for drug-induced hepatosteatosis using rat primary hepatocytes, a fluorescent lipid analog and gene expression analysis.
To evaluate new drugs' potential for hepatosteatosis, we developed a cell-based assay using a fluorescent fatty acid analog: BODIPY558/568 C12. 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 C12. The formation of fluorscecent particles or lipid droplets in the cytoplasm was confirmed by confocal laser scanning microscopy and electron microscopy respectively. The accumulation of BODIPY558/568 C12 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.
Fujimura
Hisako
H
Safety Research Laboratory, Mitsubishi Tanabe Pharma Co., 2-50 Kawagishi, 2-Chome, Toda, Saitama 335-8505, Japan. fujimura.hisako@mk.mt-pharma.co.jp
Murakami
Naoko
N
Kurabe
Michie
M
Toriumi
Wataru
W
D000818:Animals; D001896:Boron Compounds / Q000633:toxicity*; D002478:Cells, Cultured; D005234:Fatty Liver / Q000139:chemically induced* / Q000473:pathology; D005456:Fluorescent Dyes / Q000737:chemistry; D005470:Fluorometry; D015870:Gene Expression / Q000187:drug effects*; D022781:Hepatocytes / Q000187:drug effects*; D007091:Image Processing, Computer-Assisted; D008055:Lipids / Q000737:chemistry / Q000633:toxicity*; D008297:Male; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D051381:Rats; D017207:Rats, Sprague-Dawley
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D005456:Fluorescent Dyes; D008055:Lipids